Single-chain chimeric polypeptides and uses thereof

ABSTRACT

Provided herein are single-chain chimeric polypeptides that include: (i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii) a second target-binding domain. Also provided here are methods of using these single-chain chimeric polypeptides and nucleic acids encoding these single-chain chimeric polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 17/870,373, filed Jul. 21, 2022, which is a divisionalapplication of U.S. patent application Ser. No. 16/556,040, filed Aug.29, 2019, which claims priority to: U.S. Patent Application Ser. No.62/725,038, filed Aug. 30, 2018, U.S. Patent Application Ser. No.62/817,244, filed Mar. 12, 2019; U.S. Patent Application Ser. No.62/746,832, filed Oct. 17, 2018; U.S. Patent Application Ser. No.62/749,506, filed Oct. 23, 2018; U.S. Patent Application Ser. No.62/817,241, filed Mar. 12, 2019; U.S. Patent Application Ser. No.62/816,683, filed Mar. 11, 2019; and U.S. Patent Application Ser. No.62/881,039, filed Jul. 31, 2019; each of which is herein incorporated byreference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

This document includes a sequence listing in electronic format submittedto the United States Patent and Trademark Office via the electronicfiling system. The XML file, which is incorporated-by-reference herein,is titled “470390008003ST26.XML,” was created on Jul. 22, 2022, and hasa size of 180,829 bytes.

TECHNICAL FIELD

The present disclosure relates to the field of biotechnology, and morespecifically, to antigen-binding molecules.

BACKGROUND

Tissue factor (TF), a 263 amino acid integral membrane glycoprotein witha molecular weight of ˜46 kDa and the trigger protein of the extrinsicblood coagulation pathway, is the primary initiator of coagulation invivo. Tissue factor, normally not in contact with circulating blood,initiates the coagulation cascade upon exposure to the circulatingcoagulation serine protease factors. Vascular damage exposessub-endothelial cells expressing tissue factor, resulting in theformation of a calcium-dependent, high-affinity complex withpre-existing plasma factor VIIa (FVIIa). Binding of the serine proteaseFVIIa to tissue factor promotes rapid cleavage of FX to FXa and FIX toFIXa. The proteolytic activity of the resulting FXa and an activemembrane surface then inefficiently converts a small amount ofprothrombin to thrombin. The thrombin generated by FXa initiatesplatelet activation and activates minute amounts of the pro-cofactorsfactor V (FV) and factor VIII (FVIII) to become active cofactors, factorVa (FVa) and factor VIIIa (FVIIIa). FIXa complexes with FVIIIa on theplatelet surface forming the intrinsic tenase complex, which results inrapid generation of FXa. FXa complexes with FVa to form thepro-thrombinase complex on the activated platelet surface which resultsin rapid cleavage of prothrombin to thrombin.

In addition to the tissue factor-FVIIa complex, a recent study showedthat the tissue factor-FVIIa-FXa complex can activate FVIII, which wouldprovide additional levels of FVIIIa during the initiation phase. Theextrinsic pathway is paramount in initiating coagulation via theactivation of limited amounts of thrombin, whereas the intrinsic pathwaymaintains coagulation by dramatic amplification of the initial signal.

Much of the tissue factor expressed on a cell surface is “encrypted,”which must be “decrypted” for full participation in coagulation. Themechanism of “decryption” of cell-surface tissue factor is still unclearat this time, however, exposure of anionic phospholipids plays a majorrole in this process. Healthy cells actively sequester anionicphospholipids such as phosphatidyl serine (PS) to the inner leaflet ofthe plasma membrane. Following cellular damage, activation, or increasedlevels of cytosolic Ca²⁺, this bilayer asymmetry is lost, resulting inincreased PS exposure on the outer leaflet, which increases the specificactivity of cell-surface tissue factor-FVIIa complexes. PS exposure isknown to decrease the apparent Km for activation of FIX and FX by tissuefactor-FVIIa complexes, but additional mechanisms could includeconformational rearrangement of tissue factor or tissue factor-FVIIa andsubsequent exposure of substrate binding sites.

SUMMARY

The present invention is based on the discovery that soluble tissuefactor can be used as a scaffold for chimeric polypeptides including anantigen-binding domain. Based on this discovery provided herein aresingle-chain chimeric polypeptides that include: (i) a firsttarget-binding domain; (ii) a soluble tissue factor domain; and (iii) asecond target-binding domain. Also provided herein are compositions thatinclude any of the single-chain chimeric polypeptides described herein,nucleic acids that encode any of the single-chain chimeric polypeptidesdescribed herein, and cells that include any of the nucleic acids thatencode any of the single-chain chimeric polypeptides described herein.Also provided herein are methods of stimulating an immune cell andmethods of treating a subject in need thereof that include the use ofany of the single-chain chimeric polypeptides described herein. Alsoprovided herein are methods of producing any of the single-chainchimeric polypeptides described herein. Additional uses of any of thesingle-chain chimeric polypeptides are described herein.

Accordingly, provided herein is a single-chain chimeric polypeptidecomprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a second target-binding domain. In some embodiments, the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other. In some embodiments, the single-chain chimeric polypeptidefurther comprises a linker sequence between the first target-bindingdomain and the soluble tissue factor domain. In some embodiments, thesoluble tissue factor domain and the second target-binding domaindirectly abut each other. In some embodiments, the single-chain chimericpolypeptide further comprises a linker sequence between the solubletissue factor domain and the second target-binding domain. In someembodiments, the first target-binding domain and the secondtarget-binding domain directly abut each other. In some embodiments, thesingle-chain chimeric polypeptide further comprises a linker sequencebetween the first target-binding domain and the second target-bindingdomain. In some embodiments, the second target-binding domain and thesoluble tissue factor domain directly abut each other. In someembodiments, the single-chain chimeric polypeptide further comprises alinker sequence between the second target-binding domain and the solubletissue factor domain. In some embodiments, the first target-bindingdomain and the second target-binding domain bind specifically to thesame antigen. In some embodiments, the first target-binding domain andthe second target-binding domain bind specifically to the same epitope.In some embodiments, the first target-binding domain and the secondtarget-binding domain comprise the same amino acid sequence. In someembodiments, the first target-binding domain and the secondtarget-binding domain bind specifically to different antigens. In someembodiments, one or both of the first target-binding domain and thesecond target-binding domain is an antigen-binding domain. In someembodiments, the first target-binding domain and the secondtarget-binding domain are each an antigen-binding domain. In someembodiments, the antigen-binding domain comprises a scFv or a singledomain antibody. In some embodiments, one or both of the firsttarget-binding domain and the second target-binding domain bind to atarget selected from the group consisting of: CD16a, CD28, CD3, CD33,CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1,TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36,ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1,HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, aUL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155,PDGF-DD, a ligand of TGF-β receptor II (TGF-βRII), a ligand ofTGF-βRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, aligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand fora scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor forIL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, areceptor for IL-10, a receptor for IL-12, a receptor for IL-15, areceptor for IL-17, a receptor for IL-18, a receptor for IL-21, areceptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptorfor stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor forMICA, a receptor for MICB, a receptor for a ULP16-binding protein, areceptor for CD155, a receptor for CD122, and a receptor for CD28. Insome embodiments, one or both of the first target-binding domain and thesecond target-binding domain is a soluble interleukin, a solublecytokine protein, or a soluble cell surface protein. In someembodiments, the soluble interleukin, soluble cytokine protein, orsoluble cell surface protein is selected from the group consisting of:IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein. In someembodiments, one or both of the first target-binding domain and thesecond target-binding domain is a soluble interleukin receptor, asoluble cytokine receptor, or a soluble cell surface receptor. In someembodiments, the soluble interleukin receptor, soluble cytokinereceptor, or soluble cell surface receptor is a soluble TGF-β receptorII (TGF-βRII), a soluble TGF-βRIII, a soluble NKG2D, a soluble NKp30, asoluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, ascTCR, a soluble CD155, or a soluble CD28. In some embodiments, thesoluble tissue factor domain is a soluble human tissue factor domain. Insome embodiments, the soluble human tissue factor domain comprises asequence that is at least 80% identical to SEQ ID NO: 9. In someembodiments, the soluble human tissue factor domain comprises a sequencethat is at least 90% identical to SEQ ID NO: 9. In some embodiments, thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 9. In some embodiments, the single-chainchimeric polypeptide of the soluble human tissue factor domain does notcomprise one or more of: a lysine at an amino acid position thatcorresponds to amino acid position 20 of mature wildtype human tissuefactor protein; an isoleucine at an amino acid position that correspondsto amino acid position 22 of mature wildtype human tissue factorprotein; a tryptophan at an amino acid position that corresponds toamino acid position 45 of mature wildtype human tissue factor protein;an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein; atyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein; an arginineat an amino acid position that corresponds to amino acid position 135 ofmature wildtype human tissue factor protein; and a phenylalanine at anamino acid position that corresponds to amino acid position 140 ofmature wildtype human tissue factor protein. In some embodiments, thesoluble human tissue factor domain does not comprise any of: a lysine atan amino acid position that corresponds to amino acid position 20 ofmature wildtype human tissue factor protein; an isoleucine at an aminoacid position that corresponds to amino acid position 22 of maturewildtype human tissue factor protein; a tryptophan at an amino acidposition that corresponds to amino acid position 45 of mature wildtypehuman tissue factor protein; an aspartic acid at an amino acid positionthat corresponds to amino acid position 58 of mature wildtype humantissue factor protein; a tyrosine at an amino acid position thatcorresponds to amino acid position 94 of mature wildtype human tissuefactor protein; an arginine at an amino acid position that correspondsto amino acid position 135 of mature wildtype human tissue factorprotein; and a phenylalanine at an amino acid position that correspondsto amino acid position 140 of mature wildtype human tissue factorprotein. In some embodiments, the soluble tissue factor domain is notcapable of binding Factor VIIa. In some embodiments, the soluble tissuefactor domain does not convert inactive Factor X into Factor Xa. In someembodiments, the single-chain chimeric polypeptide does not stimulateblood coagulation in a mammal. In some embodiments, the single-chainchimeric polypeptide further comprises one or more additionaltarget-binding domains at its N- and/or C-terminus. In some embodiments,the single-chain chimeric polypeptide comprises one or more additionaltarget-binding domains at its N-terminus. In some embodiments, one ormore additional target-binding domains directly abuts the firsttarget-binding domain, the second target-binding domain, or the solubletissue factor domain. In some embodiments, the single-chain chimericpolypeptide further comprises a linker sequence between one of the atleast one additional target-binding domains and the first target-bindingdomain, the second target-binding domain, or the soluble tissue factordomain. In some embodiments, the single-chain chimeric polypeptidecomprises one or more additional target-binding domains at itsC-terminus. In some embodiments, one of the one or more additionaltarget-binding domains directly abuts the first target-binding domain,the second target-binding domain, or the soluble tissue factor domain.In some embodiments, the single-chain chimeric polypeptide furthercomprises a linker sequence between one of the at least one additionaltarget-binding domains and the first target-binding domain, the secondtarget-binding domain, or the soluble tissue factor domain. In someembodiments, the single-chain chimeric polypeptide comprises one or moreadditional target binding domains at its N-terminus and the C-terminus.In some embodiments, one of the one or more additional antigen bindingdomains at the N-terminus directly abuts the first target-bindingdomain, the second target-binding domain, or the soluble tissue factordomain. In some embodiments, the single-chain chimeric polypeptidefurther comprises a linker sequence between one of the one or moreadditional antigen-binding domains at the N-terminus and the firsttarget-binding domain, the second target-binding domain, or the solubletissue factor domain. In some embodiments, one of the one or moreadditional antigen binding domains at the C-terminus directly abuts thefirst target-binding domain, the second target-binding domain, or thesoluble tissue factor domain. In some embodiments, the single-chainchimeric polypeptide further comprises a linker sequence between one ofthe one or more additional antigen-binding domains at the C-terminus andthe first target-binding domain, the second target-binding domain, orthe soluble tissue factor domain. In some embodiments, two or more ofthe first target-binding domain, the second target-binding domain, andthe one or more additional target-binding domains bind specifically tothe same antigen. In some embodiments, two or more of the firsttarget-binding domain, the second target-binding domain, and the one ormore additional target-binding domains bind specifically to the sameepitope. In some embodiments, two or more of the first target-bindingdomain, the second target-binding domain, and the one or more additionaltarget-binding domains comprise the same amino acid sequence. In someembodiments, the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains each bindspecifically to the same antigen. In some embodiments, the firsttarget-binding domain, the second target-binding domain, and the one ormore additional target-binding domains each bind specifically to thesame epitope. In some embodiments, the first target-binding domain, thesecond target-binding domain, and the one or more additionaltarget-binding domains each comprise the same amino acid sequence. Insome embodiments, the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains bind specifically to different antigens. In some embodiments,one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more target-binding domains is anantigen-binding domain. In some embodiments, the first target-bindingdomain, the second target-binding domain, and the one or more additionaltarget-binding domains are each an antigen-binding domain. In someembodiments, the antigen-binding domain comprises a scFv or a singledomain antibody. In some embodiments, one or more of the firsttarget-binding domain, the second target-binding domain, and the one ormore target-binding domains bind specifically to a target selected fromthe group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22,CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3,CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200,IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA,B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR,DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-β receptorII (TGF-βRII), a ligand of TGF-βRIII, a ligand of DNAM-1, a ligand ofNKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligandfor a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptorfor IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7,a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, areceptor for IL-15, a receptor for IL-17, a receptor for IL-18, areceptor for IL-21, a receptor for PDGF-D, a receptor for stem cellfactor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand(FLT3L), a receptor for MICA, a receptor for MICB, a receptor for aULP16-binding protein, a receptor for CD155, a receptor for CD122, and areceptor for CD28. In some embodiments, one or more of the firsttarget-binding domain, the second target-binding domain, and the one ormore additional target-binding domains is a soluble interleukin orcytokine protein. In some embodiments, the soluble interleukin orcytokine protein is selected from the group consisting of: IL-1, IL-2,IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD,SCF, FLT3L, MICA, MICB, and a ULP16-binding protein. In someembodiments, one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains is a soluble interleukin or cytokine receptor. In someembodiments, the soluble receptor is a soluble TGF-β receptor II(TGF-βRII), a soluble TGF-βRIII, a soluble NKG2D, a soluble NKp30, asoluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, ascTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a solubleCD28. In some embodiments, the single-chain chimeric polypeptide furthercomprises a signal sequence at its N-terminal end. In some embodiments,the single-chain chimeric polypeptide further comprises a peptide tagpositioned at the N-terminal end or the C-terminal end of thesingle-chain chimeric polypeptide.

Also provided herein is a composition comprising any of the single-chainchimeric polypeptides discussed above. In some embodiments, thecomposition is a pharmaceutical composition. In some embodiments, thesingle-chain chimeric polypeptides comprise at least one dose of thecomposition within a kit.

Also provided herein is a method of stimulating an immune cell, themethod comprising: contacting an immune cell with an effective amount ofany of the single-chain chimeric polypeptides or compositions discussedabove. In some embodiments, the method comprises contacting the immunein vitro. In some embodiments, the method comprises obtaining the immunecell from a subject. In some embodiments, the method further comprisesobtaining the immune cell from the subject prior to the contacting step.In some embodiments, the method comprises contacting the immune cell invivo. In some embodiments, the immune cell is selected from the groupconsisting of: an immature thymocyte, a peripheral blood lymphocyte, anaïve T cell, a pluripotent Th cell precursor, a lymphoid progenitorcell, a Treg cell, a memory T cell, a Th17 cell, a Th22 cell, a Th9cell, a Th2 cell, a Th1 cell, a Th3 cell, γδ T cell, an αβ T cell, atumor-infiltrating T cell, a CD8⁺ T cell, a CD4⁺ T cell, a naturalkiller T cell, a mast cell, a macrophage, a neutrophil, a dendriticcell, a basophil, an eosinophil, and a natural killer cell. In someembodiments, the immune cell has previously been genetically modified toexpress a chimeric antigen receptor or a recombinant T-cell receptor. Insome embodiments, the method further comprises introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor after the contacting step. In someembodiments, the method further comprises administering the immune cellto a subject in need thereof. In some embodiments, the method comprisesidentifying or diagnosing the subject as having an age-related diseaseor condition. In some embodiments, the age-related disease or conditionis selected from the group consisting of: Alzheimer's disease, aneurysm,cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension,idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction. In some embodiments, themethod comprises identifying or diagnosing the subject as having acancer. In some embodiments, the cancer is selected from the groupconsisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma. In some embodiments,the method comprises diagnosing or identifying the subject as having aninfectious disease. In some embodiments, the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Also provided herein is a method of inducing or increasing proliferationof an immune cell, the method comprising: contacting an immune cell withan effective amount of any of the single-chain chimeric polypeptides orcompositions discussed herein. In some embodiments, the method comprisescontacting the immune cell in vitro. In some embodiments, the methodcomprises obtaining the immune cell from a subject. In some embodiments,method further comprises obtaining the immune cell from the subjectprior to the contacting step. In some embodiments, the method comprisescontacting the immune cell in vivo. In some embodiments, the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ T cell, aCD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell. In some embodiments, the immune cell has previously beengenetically modified to express a chimeric antigen receptor or arecombinant T-cell receptor. In some embodiments, the method furthercomprises, after the contacting step, introducing into the immune cell anucleic acid encoding a chimeric antigen-receptor or a recombinantT-cell receptor. In some embodiments, the method further comprisesadministering the immune cell to a subject in need thereof. In someembodiments, the method comprises identifying or diagnosing the subjectas having an age-related disease or condition. In some embodiments, theage-related disease or condition is selected from the group consistingof: Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction. In some embodiments,the method comprises identifying or diagnosing the subject as having acancer. In some embodiments, the cancer is selected from the groupconsisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma. In some embodiments,the method comprises diagnosing or identifying the subject as having aninfectious disease. In some embodiments, the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Also provided herein is a method of inducing differentiation of animmune cell into a memory or memory-like immune cell, the methodcomprising: contacting an immune cell with an effective amount of any ofthe single-chain chimeric polypeptides or compositions discussed above.In some embodiments, the method comprises contacting the immune cell invitro. In some embodiments, the method comprises obtaining the immunecell from a subject. In some embodiments, the method further comprisesobtaining the immune cell from the subject prior to the contacting step.In some embodiments, the method comprises contacting the immune cell invivo. In some embodiments, the immune cell is selected from the groupconsisting of: an immature thymocyte, a peripheral blood lymphocyte, anaïve T cell, a pluripotent Th cell precursor, a lymphoid progenitorcell, a Treg cell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2 cell, aTh1 cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating Tcell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mastcell, a macrophage, a neutrophil, a dendritic cell, a basophil, aneosinophil, and a natural killer cell. In some embodiments, the methodcomprises genetically modifying the immune cell to express a chimericantigen receptor or a recombinant T-cell receptor. In some embodiments,the method further comprises introducing into the immune cell a nucleicacid encoding a chimeric antigen-receptor or a recombinant T-cellreceptor after the contacting step. In some embodiments, the methodfurther comprises administering the immune cell to a subject in needthereof. In some embodiments, the method comprises identifying ordiagnosing the subject as having an age-related disease or condition. Insome embodiments, the age-related disease or condition is selected fromthe group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis,fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonaryfibrosis, inflammatory bowel disease, intervertebral disc degeneration,macular degeneration, osteoarthritis, type 2 diabetes mellitus, adiposeatrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathicpulmonary fibrosis, kidney transplant failure, liver fibrosis, loss ofbone mass, myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction. In some embodiments,the method comprises identifying or diagnosing the subject as having acancer. In some embodiments, the cancer is selected from the groupconsisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma. In some embodiments,the method comprises diagnosing or identifying the subject as having aninfectious disease. In some embodiments, the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Also provided herein is a method of killing a cancer cell, an infectedcell, or a senescent cell in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of any of the single-chain chimeric polypeptides or compositionsdiscussed above. In some embodiments, the method comprises identifyingor diagnosing the subject as having a cancer. In some embodiments, thecancer is selected from the group consisting of: solid tumor,hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma,melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cellneoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acutemyeloid leukemia (AML), chronic myeloid leukemia (CML), acutelymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneousT-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma,lung cancer, renal cell carcinoma, gastric and esophageal cancer,pancreatic cancer, prostate cancer, breast cancer, colorectal cancer,ovarian cancer, non-small cell lung carcinoma, squamous cell head andneck carcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma. In some embodiments, the method identifying ordiagnosing the subject as having an aging-related disease or condition.In some embodiments, the aging-related disease or condition is selectedfrom the group consisting of: Alzheimer's disease, aneurysm, cysticfibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathicpulmonary fibrosis, inflammatory bowel disease, intervertebral discdegeneration, macular degeneration, osteoarthritis, type 2 diabetesmellitus, adipose atrophy, lipodystrophy, atherosclerosis, cataracts,COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liverfibrosis, loss of bone mass, myocardial infarction, sarcopenia, woundhealing, alopecia, cardiomyocyte hypertrophy, osteoarthritis,Parkinson's disease, age-associated loss of lung tissue elasticity,macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis,NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington'sdisease, spinocerebellar ataxia, multiple sclerosis, and renaldysfunction.

Also provided herein is a method of treating a subject in need thereof,the method comprising administering to the subject a therapeuticallyeffective amount of any of the single-chain chimeric polypeptides orcompositions discussed above. In some embodiments, the method comprisesidentifying or diagnosing the subject as having a cancer. In someembodiments, the cancer is selected from the group consisting of: solidtumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma,neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma,B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cellnon-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia(CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML),acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS),cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelialcarcinoma, lung cancer, renal cell carcinoma, gastric and esophagealcancer, pancreatic cancer, prostate cancer, breast cancer, colorectalcancer, ovarian cancer, non-small cell lung carcinoma, squamous cellhead and neck carcinoma, endometrial cancer, cervical cancer, livercancer, and hepatocellular carcinoma. In some embodiments, the methodcomprises identifying or diagnosing the subject as having anaging-related disease or condition. In some embodiments, theaging-related disease or condition is selected from the group consistingof: Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction. In some embodiments,the method comprises diagnosing or identifying the subject as having aninfectious disease. In some embodiments, the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Also provided herein are nucleic acids encoding any of the single-chainchimeric polypeptides discussed above. Some embodiments include a vectorcomprising the nucleic acid discussed above. In some embodiments, thevector is an expression vector. Some embodiments include a cellcomprising the nucleic acid or the vector discussed above.

Also provided herein is a method of producing a single-chain chimericpolypeptide, the method comprising: culturing the cell in a culturemedium under conditions sufficient to result in the production of thesingle-chain chimeric polypeptide; and recovering the single-chainchimeric polypeptide from the cell and/or the culture medium. Someembodiments comprise single-chain chimeric polypeptide produced by thediscussed above. In some embodiments of any of the single-chain chimericpolypeptides provided herein, the human soluble tissue factor domaindoes not initiate blood coagulation. In some embodiments of any of thesingle-chain chimeric polypeptides provided herein, the soluble tissuefactor domain comprises or consists of a soluble wildtype human tissuefactor.

In some embodiments, the mutant soluble human tissue factor domaincomprises a sequence that is at least 80% identical to SEQ ID NO: 96. Insome embodiments, the soluble human tissue factor domain comprises asequence that is at least 90% identical to SEQ ID NO: 96. In someembodiments, the soluble human tissue factor domain comprises a sequencethat is at least 95% identical to SEQ ID NO: 96. In some embodiments,the soluble human tissue factor domain comprises a sequence that is 100%identical to SEQ ID NO: 96. In some embodiments, the soluble humantissue factor domain comprises a sequence that is at least 80% identicalto SEQ ID NO: 97. In some embodiments, the soluble human tissue factordomain comprises a sequence that is at least 90% identical to SEQ ID NO:97. In some embodiments, the soluble human tissue factor domaincomprises a sequence that is at least 95% identical to SEQ ID NO: 97. Insome embodiments, the soluble human tissue factor domain comprises asequence that is 100% identical to SEQ ID NO: 97.

As used herein, the term “chimeric” refers to a polypeptide thatincludes amino acid sequences (e.g., domains) originally derived fromtwo different sources (e.g., two different naturally-occurring proteins,e.g., from the same or different species). For example, a chimericpolypeptide can include domains from at least two different naturallyoccurring human proteins. In some examples, a chimeric polypeptide caninclude a domain that is a synthetic sequence (e.g., a scFv) and adomain that is derived from a naturally-occurring protein (e.g., anaturally-occurring human protein). In some embodiments, a chimericpolypeptide can include at least two different domains that aresynthetic sequences (e.g., two different scFvs).

An “antigen-binding domain” is one or more protein domain(s) (e.g.,formed from amino acids from a single polypeptide or formed from aminoacids from two or more polypeptides (e.g., the same or differentpolypeptides) that is capable of specifically binding to one or moredifferent antigen(s). In some examples, an antigen-binding domain canbind to an antigen or epitope with specificity and affinity similar tothat of naturally-occurring antibodies. In some embodiments, theantigen-binding domain can be an antibody or a fragment thereof. In someembodiments, an antigen-binding domain can include an alternativescaffold. Non-limiting examples of antigen-binding domains are describedherein. Additional examples of antigen-binding domains are known in theart.

A “soluble tissue factor domain” refers to a polypeptide having at least70% identity (e.g., at least 75% identity, at least 80% identity, atleast 85% identity, at least 90% identity, at least 95% identity, atleast 99% identity, or 100% identical) to a segment of a wildtypemammalian tissue factor protein (e.g., a wildtype human tissue factorprotein) that lacks the transmembrane domain and the intracellulardomain. Non-limiting examples of soluble tissue factor domains aredescribed herein.

The term “soluble interleukin protein” is used herein to refer to amature and secreted interleukin protein or a biologically activefragment thereof. In some examples, a soluble interleukin protein caninclude a sequence that is at least 70% identical, at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, at least 95% identical, at least 99% identical, or 100%identical to a wildtype mature and secreted mammalian interleukinprotein (e.g., a wildtype human interleukin protein) and retains itsbiological activity. Non-limiting examples of soluble interleukinproteins are described herein.

The term “soluble cytokine protein” is used herein to refer to a matureand secreted cytokine protein or a biologically active fragment thereof.In some examples, a soluble cytokine protein can include a sequence thatis at least 70% identical, at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 99% identical, or 100% identical to a wildtypemature and secreted mammalian interleukin protein (e.g., a wildtypehuman interleukin protein) and retains its biological activity.Non-limiting examples of soluble cytokine proteins are described herein.

The term “soluble interleukin receptor” is used herein in the broadestsense to refer to a polypeptide that lacks a transmembrane domain (andoptionally an intracellular domain) that is capable of binding one ormore of its natural ligands (e.g., under physiological conditions, e.g.,in phosphate buffered saline at room temperature). For example, asoluble interleukin receptor can include a sequence that is at least 70%identical (e.g., at least 75% identical, at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical, atleast 99% identical, or 100% identical) to an extracellular domain ofwildtype interleukin receptor and retains its ability to specificallybind to one or more of its natural ligands, but lacks its transmembranedomain (and optionally, further lacks its intracellular domain).Non-limiting examples of soluble interleukin receptors are describedherein.

The term “soluble cytokine receptor” is used herein in the broadestsense to refer to a polypeptide that lacks a transmembrane domain (andoptionally an intracellular domain) that is capable of binding one ormore of its natural ligands (e.g., under physiological conditions, e.g.,in phosphate buffered saline at room temperature). For example, asoluble cytokine receptor can include a sequence that is at least 70%identical (e.g., at least 75% identical, at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical, atleast 99% identical, or 100% identical) to an extracellular domain ofwildtype cytokine receptor and retains its ability to specifically bindto one or more of its natural ligands, but lacks its transmembranedomain (and optionally, further lacks its intracellular domain).Non-limiting examples of soluble cytokine receptors are describedherein.

The term “antibody” is used herein in its broadest sense and includescertain types of immunoglobulin molecules that include one or moreantigen-binding domains that specifically bind to an antigen or epitope.An antibody specifically includes, e.g., intact antibodies (e.g., intactimmunoglobulins), antibody fragments, and multi-specific antibodies. Oneexample of an antigen-binding domain is an antigen-binding domain formedby a VH-VL dimer. Additional examples of an antibody are describedherein. Additional examples of an antibody are known in the art.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between an antigen-binding site and its binding partner(e.g., an antigen or epitope). Unless indicated otherwise, as usedherein, “affinity” refers to intrinsic binding affinity, which reflectsa 1:1 interaction between members of an antigen-binding domain and anantigen or epitope. The affinity of a molecule X for its partner Y canbe represented by the dissociation equilibrium constant (K_(D)). Thekinetic components that contribute to the dissociation equilibriumconstant are described in more detail below. Affinity can be measured bycommon methods known in the art, including those described herein.Affinity can be determined, for example, using surface plasmon resonance(SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g.,FORTEBIO®). Additional methods for determining the affinity for anantigen-binding domain and its corresponding antigen or epitope areknown in the art.

A “single-chain polypeptide” as used herein to refers to a singleprotein chain. The term “pair of affinity domains” is two differentprotein domain(s) that bind specifically to each other with a K_(D) ofless than of less than 1×10⁻⁷ M (e.g., less than 1×10⁻⁸ M, less than1×10⁻⁹ M, less than 1×10⁻¹⁰ M, or less than 1×10⁻¹¹ M). In someexamples, a pair of affinity domains can be a pair ofnaturally-occurring proteins. In some embodiments, a pair of affinitydomains can be a pair of synthetic proteins. Non-limiting examples ofpairs of affinity domains are described herein.

The term “epitope” means a portion of an antigen that specifically bindsto an antigen-binding domain. Epitopes can, e.g., consist ofsurface-accessible amino acid residues and/or sugar side chains and mayhave specific three-dimensional structural characteristics, as well asspecific charge characteristics. Conformational and non-conformationalepitopes are distinguished in that the binding to the former but not thelatter may be lost in the presence of denaturing solvents. An epitopemay comprise amino acid residues that are directly involved in thebinding, and other amino acid residues, which are not directly involvedin the binding. Methods for identifying an epitope to which anantigen-binding domain binds are known in the art.

An “immune effector cell” refers to a cell of the immune system of amammal that is capable, directly or indirectly, of recognizing and/orcausing cytostasis or cell death of a pathogenic cell (e.g., a cancercell) in the mammal. Non-limiting examples of immune effector cellsinclude macrophages, T-lymphocytes (e.g., cytotoxic T-lymphocytes andT-helper cells), natural killer cells, neutrophils, monocytes, andeosinophils. Additional examples of immune effector cells are known inthe art.

The term “treatment” means to ameliorate at least one symptom of adisorder. In some examples, the disorder being treated is cancer and toameliorate at least one symptom of cancer includes reducing aberrantproliferation, gene expression, signaling, translation, and/or secretionof factors. Generally, the methods of treatment include administering atherapeutically effective amount of composition that reduces at leastone symptom of a disorder to a subject who is in need of, or who hasbeen determined to be in need of such treatment.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 are schematic diagrams of an exemplary αCD3scFv/TF/αCD28scFvsingle-chain chimeric polypeptide.

FIG. 2 is a chromatograph showing the elution of an exemplaryαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide from ananti-tissue factor antibody affinity column.

FIG. 3 is a chromatograph showing the elution of a Superdex 200 Increase10/300 GL gel filtration column loaded with an exemplaryαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide.

FIG. 4 is a sodium dodecyl sulfate polyacrylamide gel (4-12% NuPageBis-Tris gel) of an exemplary αCD3scFv/TF/αCD28scFv single-chainchimeric polypeptide purified using an anti-tissue factor antibodyaffinity column.

FIG. 5 is a graph showing the ELISA quantitation of an exemplaryαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide performed usingthe methods described in Example 1. Purified tissue factor was used asthe control.

FIG. 6 is a graph showing the ability of an exemplaryαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide to stimulateCD25 expression in CD4⁺ T-cells isolated from blood from two donors. Theexperiments were performed as described in Example 2.

FIG. 7 is a graph showing the ability of an exemplaryαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide to stimulateCD25 expression in CD8⁺ T-cells isolated from blood from two donors. Theexperiments were performed as described in Example 2.

FIG. 8 is a graph showing the ability of an exemplaryαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide to stimulateCD69 expression in CD4⁺ T-cells isolated from blood from two donors. Theexperiments were performed as described in Example 2.

FIG. 9 are schematic diagrams of an exemplary IL-2/TF/IL-2 single-chainchimeric polypeptide.

FIG. 10 shows IL-2 activity in IL-2/TF/IL-2 as compared to recombinantIL-2 using a 32Dβ cell proliferation assay.

FIG. 11 shows IL-2 activity in IL-2/TF/IL-2 as compared to recombinantIL-2 using a CTLL-2 cell proliferation assay.

FIG. 12 shows the fasting blood glucose levels in ApoE^(−/−) mice fedwith standard chow or a high fat diet and treated with a PBS control(untreated) or with IL-2/TF/IL-2.

FIG. 13 shows the ratio of CD4⁺CD25⁺FoxP3⁺ T regulatory cells in bloodlymphocytes from ApoE−/− mice fed with standard chow or a high fat dietand treated with a PBS control (untreated) or with IL-2/TF/IL-2.

FIG. 14 is a line graph showing the chromatographic profile ofIL-2/TF/IL-2 protein containing cell culture supernatant followingbinding and elution on anti-TF antibody resin.

FIG. 15 shows an analytical SEC profile of IL-2/TF/IL-2.

FIGS. 16A and 16B show reduced SDS-PAGE analysis of IL-2/TF/IL-2 beforeand after deglycosylation. FIG. 16A shows reduced SDS-PAGE analysis ofIL-2/TF/IL-2 before deglycosylation. FIG. 16B shows reduced SDS-PAGEanalysis of IL-2/TF/IL-2 after deglycosylation.

FIGS. 17A and 17B show results of immunostimulation in C57BL/6 miceusing IL-2/TF/IL-2. FIG. 17A shows spleen weight following treatmentwith IL-2/TF/IL-2. FIG. 17B shows the percentages of immune cell typesfollowing IL-2/TF/IL-2 treatment.

FIG. 18 shows upregulation of CD25 expression of CD4⁺ T cells in micetreated with IL-2/TF/IL-2.

FIG. 19 shows the pharmacokinetics of IL-2/TF/IL-2 in C57BL/6 mice.

FIGS. 20A and 20B show effects of IL-2/TF/IL-2 in attenuating theformation of high fat-induced atherosclerotic plaques in ApoE^(−/−)mice. FIG. 20A shows a representative view of atherosclerotic plaquesfrom ApoE^(−/−) mice fed with standard chow or a high fat diet andtreated with either PBS control or IL-2/TF/IL-2. FIG. 20B shows theresults of quantitative analysis of atherosclerotic plaques of eachgroup.

FIG. 21 shows fasting glucose levels in IL-2/TF/IL-2 treated-mice ascompared to control-treated mice.

FIG. 22 shows the percentage of CD4⁺CD25⁺FoxP3⁺ Tregs in bloodlymphocytes from mice treated with IL-2/TF/IL-2 and control-treatedmice.

FIG. 23 are schematic diagrams of an exemplary IL-15/TF/IL-15single-chain chimeric polypeptide.

FIG. 24 shows the IL-15 activity of IL-15/TF/IL-15 as compared torecombinant IL-15 in a 32Dβ cell proliferation assay.

FIG. 25 is a line graph showing the chromatographic profile ofIL-15/TF/IL-15 protein containing cell culture supernatant followingbinding and elution on anti-TF antibody resin.

FIGS. 26A and 26B show reduced SDS-PAGE analysis of IL-15/TF/IL-15before and after deglycosylation. FIG. 26A shows reduced SDS-PAGEanalysis of IL-15/TF/IL-15 before deglycosylation. FIG. 26B showsreduced SDS-PAGE analysis of IL-15/TF/IL-15 after deglycosylation.

FIGS. 27A-27C is a set of graphs showing immunostimulation in C57BL/6mice following treatment with 2t2.

FIGS. 28A-28C is a set of graphs showing in vivo stimulation of Tregs,NK cells, and CD8⁺ T cells in ApoE^(−/−) mice fed with a Western dietand treated with 2t2.

FIGS. 29A and 29B is a set of graphs showing induction of splenocyteproliferation by 2t2 in C57BL/6 mice.

FIGS. 30A and 30B is a set of graphs showing in vivo induction ofproliferation of NK cells and CD8⁺ T cells in ApoE^(−/−) mice fed with aWestern diet and treated with 2t2.

FIGS. 31A-31C is a set of graphs showing amelioration of the Westerndiet-induced hyperglycemia in ApoE^(−/−) mice by 2t2.

FIG. 32 shows upregulation of CD44 memory T cells upon treatment with2t2.

FIG. 33 shows a graph of Factor X (FX) activation following treatmentwith single-chain or multi-chain chimeric polypeptides.

FIG. 34A-34C show human blood lymphocyte pStat5a responses inCD4⁺CD25^(hi)T_(reg) cells, CD4⁺CD25⁻T_(con) cells, or in CD8⁺ T_(con)cells in response to 2t2 or IL2 treatment. FIG. 34A shows pSTAT5responses in CD4⁺ CD25^(hi)T_(reg) cells. FIG. 34B shows pSTAT5responses in CD4⁺CD25⁻ T_(con) cells. FIG. 34C shows pSTAT5 responses inCD8⁺ T_(con) cells.

DETAILED DESCRIPTION

Provided herein are single-chain chimeric polypeptides that include: (i)a first target-binding domain (e.g., any of the target-binding domainsdescribed herein or known in the art), (ii) a soluble tissue factordomain (e.g., any of the exemplary soluble tissue factor domainsdescribed herein or known in the art), and (iii) as secondtarget-binding domain (e.g., any of the target-binding domains describedherein or known in the art).

Also provided herein are compositions that include any of thesingle-chain chimeric polypeptides described herein, nucleic acids thatencode any of the single-chain chimeric polypeptides described herein,and cells that include any of the nucleic acids that encode any of thesingle-chain chimeric polypeptides described herein. Also providedherein are methods of stimulating an immune cell and methods of treatinga subject in need thereof that include the use of any of thesingle-chain chimeric polypeptides described herein. Also providedherein are methods of producing any of the single-chain chimericpolypeptides described herein.

In some examples of any of the single-chain chimeric polypeptidesdescribed herein, the single-chain chimeric polypeptide can have a totallength of about 50 amino acids to about 3000 amino acids, about 50 aminoacids to about 2500 amino acids, about 50 amino acids to about 2000amino acids, about 50 amino acids to about 1500 amino acids, about 50amino acids to about 1000 amino acids, about 50 amino acids to about 950amino acids, about 50 amino acids to about 900 amino acids, about 50amino acids to about 850 amino acids, about 50 amino acids to about 800amino acids, about 50 amino acids to about 750 amino acids, about 50amino acids to about 700 amino acids, about 50 amino acids to about 650amino acids, about 50 amino acids to about 600 amino acids, about 50amino acids to about 550 amino acids, about 50 amino acids to about 500amino acids, about 50 amino acids to about 480 amino acids, about 50amino acids to about 460 amino acids, about 50 amino acids to about 440amino acids, about 50 amino acids to about 420 amino acids, about 50amino acids to about 400 amino acids, about 50 amino acids to about 380amino acids, about 50 amino acids to about 360 amino acids, about 50amino acids to about 340 amino acids, about 50 amino acids to about 320amino acids, about 50 amino acids to about 300 amino acids, about 50amino acids to about 280 amino acids, about 50 amino acids to about 260amino acids, about 50 amino acids to about 240 amino acids, about 50amino acids to about 220 amino acids, about 50 amino acids to about 200amino acids, about 50 amino acids to about 150 amino acids, about 50amino acids to about 100 amino acids, about 100 amino acids to about3000 amino acids, about 100 amino acids to about 2500 amino acids, about100 amino acids to about 2000 amino acids, about 100 amino acids toabout 1500 amino acids, about 100 amino acids to about 1000 amino acids,about 100 amino acids to about 950 amino acids, about 100 amino acids toabout 900 amino acids, about 100 amino acids to about 850 amino acids,about 100 amino acids to about 800 amino acids, about 100 amino acids toabout 750 amino acids, about 100 amino acids to about 700 amino acids,about 100 amino acids to about 650 amino acids, about 100 amino acids toabout 600 amino acids, about 100 amino acids to about 550 amino acids,about 100 amino acids to about 500 amino acids, about 100 amino acids toabout 480 amino acids, about 100 amino acids to about 460 amino acids,about 100 amino acids to about 440 amino acids, about 100 amino acids toabout 420 amino acids, about 100 amino acids to about 400 amino acids,about 100 amino acids to about 380 amino acids, about 100 amino acids toabout 360 amino acids, about 100 amino acids to about 340 amino acids,about 100 amino acids to about 320 amino acids, about 100 amino acids toabout 300 amino acids, about 100 amino acids to about 280 amino acids,about 100 amino acids to about 260 amino acids, about 100 amino acids toabout 240 amino acids, about 100 amino acids to about 220 amino acids,about 100 amino acids to about 200 amino acids, about 100 amino acids toabout 150 amino acids, about 150 amino acids to about 3000 amino acids,about 150 amino acids to about 2500 amino acids, about 150 amino acidsto about 2000 amino acids, about 150 amino acids to about 1500 aminoacids, about 150 amino acids to about 1000 amino acids, about 150 aminoacids to about 950 amino acids, about 150 amino acids to about 900 aminoacids, about 150 amino acids to about 850 amino acids, about 150 aminoacids to about 800 amino acids, about 150 amino acids to about 750 aminoacids, about 150 amino acids to about 700 amino acids, about 150 aminoacids to about 650 amino acids, about 150 amino acids to about 600 aminoacids, about 150 amino acids to about 550 amino acids, about 150 aminoacids to about 500 amino acids, about 150 amino acids to about 480 aminoacids, about 150 amino acids to about 460 amino acids, about 150 aminoacids to about 440 amino acids, about 150 amino acids to about 420 aminoacids, about 150 amino acids to about 400 amino acids, about 150 aminoacids to about 380 amino acids, about 150 amino acids to about 360 aminoacids, about 150 amino acids to about 340 amino acids, about 150 aminoacids to about 320 amino acids, about 150 amino acids to about 300 aminoacids, about 150 amino acids to about 280 amino acids, about 150 aminoacids to about 260 amino acids, about 150 amino acids to about 240 aminoacids, about 150 amino acids to about 220 amino acids, about 150 aminoacids to about 200 amino acids, about 200 amino acids to about 3000amino acids, about 200 amino acids to about 2500 amino acids, about 200amino acids to about 2000 amino acids, about 200 amino acids to about1500 amino acids, about 200 amino acids to about 1000 amino acids, about200 amino acids to about 950 amino acids, about 200 amino acids to about900 amino acids, about 200 amino acids to about 850 amino acids, about200 amino acids to about 800 amino acids, about 200 amino acids to about750 amino acids, about 200 amino acids to about 700 amino acids, about200 amino acids to about 650 amino acids, about 200 amino acids to about600 amino acids, about 200 amino acids to about 550 amino acids, about200 amino acids to about 500 amino acids, about 200 amino acids to about480 amino acids, about 200 amino acids to about 460 amino acids, about200 amino acids to about 440 amino acids, about 200 amino acids to about420 amino acids, about 200 amino acids to about 400 amino acids, about200 amino acids to about 380 amino acids, about 200 amino acids to about360 amino acids, about 200 amino acids to about 340 amino acids, about200 amino acids to about 320 amino acids, about 200 amino acids to about300 amino acids, about 200 amino acids to about 280 amino acids, about200 amino acids to about 260 amino acids, about 200 amino acids to about240 amino acids, about 200 amino acids to about 220 amino acids, about220 amino acids to about 3000 amino acids, about 220 amino acids toabout 2500 amino acids, about 220 amino acids to about 2000 amino acids,about 220 amino acids to about 1500 amino acids, about 220 amino acidsto about 1000 amino acids, about 220 amino acids to about 950 aminoacids, about 220 amino acids to about 900 amino acids, about 220 aminoacids to about 850 amino acids, about 220 amino acids to about 800 aminoacids, about 220 amino acids to about 750 amino acids, about 220 aminoacids to about 700 amino acids, about 220 amino acids to about 650 aminoacids, about 220 amino acids to about 600 amino acids, about 220 aminoacids to about 550 amino acids, about 220 amino acids to about 500 aminoacids, about 220 amino acids to about 480 amino acids, about 220 aminoacids to about 460 amino acids, about 220 amino acids to about 440 aminoacids, about 220 amino acids to about 420 amino acids, about 220 aminoacids to about 400 amino acids, about 220 amino acids to about 380 aminoacids, about 220 amino acids to about 360 amino acids, about 220 aminoacids to about 340 amino acids, about 220 amino acids to about 320 aminoacids, about 220 amino acids to about 300 amino acids, about 220 aminoacids to about 280 amino acids, about 220 amino acids to about 260 aminoacids, about 220 amino acids to about 240 amino acids, about 240 aminoacids to about 3000 amino acids, about 240 amino acids to about 2500amino acids, about 240 amino acids to about 2000 amino acids, about 240amino acids to about 1500 amino acids, about 240 amino acids to about1000 amino acids, about 240 amino acids to about 950 amino acids, about240 amino acids to about 900 amino acids, about 240 amino acids to about850 amino acids, about 240 amino acids to about 800 amino acids, about240 amino acids to about 750 amino acids, about 240 amino acids to about700 amino acids, about 240 amino acids to about 650 amino acids, about240 amino acids to about 600 amino acids, about 240 amino acids to about550 amino acids, about 240 amino acids to about 500 amino acids, about240 amino acids to about 480 amino acids, about 240 amino acids to about460 amino acids, about 240 amino acids to about 440 amino acids, about240 amino acids to about 420 amino acids, about 240 amino acids to about400 amino acids, about 240 amino acids to about 380 amino acids, about240 amino acids to about 360 amino acids, about 240 amino acids to about340 amino acids, about 240 amino acids to about 320 amino acids, about240 amino acids to about 300 amino acids, about 240 amino acids to about280 amino acids, about 240 amino acids to about 260 amino acids, about260 amino acids to about 3000 amino acids, about 260 amino acids toabout 2500 amino acids, about 260 amino acids to about 2000 amino acids,about 260 amino acids to about 1500 amino acids, about 260 amino acidsto about 1000 amino acids, about 260 amino acids to about 950 aminoacids, about 260 amino acids to about 900 amino acids, about 260 aminoacids to about 850 amino acids, about 260 amino acids to about 800 aminoacids, about 260 amino acids to about 750 amino acids, about 260 aminoacids to about 700 amino acids, about 260 amino acids to about 650 aminoacids, about 260 amino acids to about 600 amino acids, about 260 aminoacids to about 550 amino acids, about 260 amino acids to about 500 aminoacids, about 260 amino acids to about 480 amino acids, about 260 aminoacids to about 460 amino acids, about 260 amino acids to about 440 aminoacids, about 260 amino acids to about 420 amino acids, about 260 aminoacids to about 400 amino acids, about 260 amino acids to about 380 aminoacids, about 260 amino acids to about 360 amino acids, about 260 aminoacids to about 340 amino acids, about 260 amino acids to about 320 aminoacids, about 260 amino acids to about 300 amino acids, about 260 aminoacids to about 280 amino acids, about 280 amino acids to about 3000amino acids, about 280 amino acids to about 2500 amino acids, about 280amino acids to about 2000 amino acids, about 280 amino acids to about1500 amino acids, about 280 amino acids to about 1000 amino acids, about280 amino acids to about 950 amino acids, about 280 amino acids to about900 amino acids, about 280 amino acids to about 850 amino acids, about280 amino acids to about 800 amino acids, about 280 amino acids to about750 amino acids, about 280 amino acids to about 700 amino acids, about280 amino acids to about 650 amino acids, about 280 amino acids to about600 amino acids, about 280 amino acids to about 550 amino acids, about280 amino acids to about 500 amino acids, about 280 amino acids to about480 amino acids, about 280 amino acids to about 460 amino acids, about280 amino acids to about 440 amino acids, about 280 amino acids to about420 amino acids, about 280 amino acids to about 400 amino acids, about280 amino acids to about 380 amino acids, about 280 amino acids to about360 amino acids, about 280 amino acids to about 340 amino acids, about280 amino acids to about 320 amino acids, about 280 amino acids to about300 amino acids, about 300 amino acids to about 3000 amino acids, about300 amino acids to about 2500 amino acids, about 300 amino acids toabout 2000 amino acids, about 300 amino acids to about 1500 amino acids,about 300 amino acids to about 1000 amino acids, about 300 amino acidsto about 950 amino acids, about 300 amino acids to about 900 aminoacids, about 300 amino acids to about 850 amino acids, about 300 aminoacids to about 800 amino acids, about 300 amino acids to about 750 aminoacids, about 300 amino acids to about 700 amino acids, about 300 aminoacids to about 650 amino acids, about 300 amino acids to about 600 aminoacids, about 300 amino acids to about 550 amino acids, about 300 aminoacids to about 500 amino acids, about 300 amino acids to about 480 aminoacids, about 300 amino acids to about 460 amino acids, about 300 aminoacids to about 440 amino acids, about 300 amino acids to about 420 aminoacids, about 300 amino acids to about 400 amino acids, about 300 aminoacids to about 380 amino acids, about 300 amino acids to about 360 aminoacids, about 300 amino acids to about 340 amino acids, about 300 aminoacids to about 320 amino acids, about 320 amino acids to about 3000amino acids, about 320 amino acids to about 2500 amino acids, about 320amino acids to about 2000 amino acids, about 320 amino acids to about1500 amino acids, about 320 amino acids to about 1000 amino acids, about320 amino acids to about 950 amino acids, about 320 amino acids to about900 amino acids, about 320 amino acids to about 850 amino acids, about320 amino acids to about 800 amino acids, about 320 amino acids to about750 amino acids, about 320 amino acids to about 700 amino acids, about320 amino acids to about 650 amino acids, about 320 amino acids to about600 amino acids, about 320 amino acids to about 550 amino acids, about320 amino acids to about 500 amino acids, about 320 amino acids to about480 amino acids, about 320 amino acids to about 460 amino acids, about320 amino acids to about 440 amino acids, about 320 amino acids to about420 amino acids, about 320 amino acids to about 400 amino acids, about320 amino acids to about 380 amino acids, about 320 amino acids to about360 amino acids, about 320 amino acids to about 340 amino acids, about340 amino acids to about 3000 amino acids, about 340 amino acids toabout 2500 amino acids, about 340 amino acids to about 2000 amino acids,about 340 amino acids to about 1500 amino acids, about 340 amino acidsto about 1000 amino acids, about 340 amino acids to about 950 aminoacids, about 340 amino acids to about 900 amino acids, about 340 aminoacids to about 850 amino acids, about 340 amino acids to about 800 aminoacids, about 340 amino acids to about 750 amino acids, about 340 aminoacids to about 700 amino acids, about 340 amino acids to about 650 aminoacids, about 340 amino acids to about 600 amino acids, about 340 aminoacids to about 550 amino acids, about 340 amino acids to about 500 aminoacids, about 340 amino acids to about 480 amino acids, about 340 aminoacids to about 460 amino acids, about 340 amino acids to about 440 aminoacids, about 340 amino acids to about 420 amino acids, about 340 aminoacids to about 400 amino acids, about 340 amino acids to about 380 aminoacids, about 340 amino acids to about 360 amino acids, about 360 aminoacids to about 3000 amino acids, about 360 amino acids to about 2500amino acids, about 360 amino acids to about 2000 amino acids, about 360amino acids to about 1500 amino acids, about 360 amino acids to about1000 amino acids, about 360 amino acids to about 950 amino acids, about360 amino acids to about 900 amino acids, about 360 amino acids to about850 amino acids, about 360 amino acids to about 800 amino acids, about360 amino acids to about 750 amino acids, about 360 amino acids to about700 amino acids, about 360 amino acids to about 650 amino acids, about360 amino acids to about 600 amino acids, about 360 amino acids to about550 amino acids, about 360 amino acids to about 500 amino acids, about360 amino acids to about 480 amino acids, about 360 amino acids to about460 amino acids, about 360 amino acids to about 440 amino acids, about360 amino acids to about 420 amino acids, about 360 amino acids to about400 amino acids, about 360 amino acids to about 380 amino acids, about380 amino acids to about 3000 amino acids, about 380 amino acids toabout 2500 amino acids, about 380 amino acids to about 2000 amino acids,about 380 amino acids to about 1500 amino acids, about 380 amino acidsto about 1000 amino acids, about 380 amino acids to about 950 aminoacids, about 380 amino acids to about 900 amino acids, about 380 aminoacids to about 850 amino acids, about 380 amino acids to about 800 aminoacids, about 380 amino acids to about 750 amino acids, about 380 aminoacids to about 700 amino acids, about 380 amino acids to about 650 aminoacids, about 380 amino acids to about 600 amino acids, about 380 aminoacids to about 550 amino acids, about 380 amino acids to about 500 aminoacids, about 380 amino acids to about 480 amino acids, about 380 aminoacids to about 460 amino acids, about 380 amino acids to about 440 aminoacids, about 380 amino acids to about 420 amino acids, about 380 aminoacids to about 400 amino acids, about 400 amino acids to about 3000amino acids, about 400 amino acids to about 2500 amino acids, about 400amino acids to about 2000 amino acids, about 400 amino acids to about1500 amino acids, about 400 amino acids to about 1000 amino acids, about400 amino acids to about 950 amino acids, about 400 amino acids to about900 amino acids, about 400 amino acids to about 850 amino acids, about400 amino acids to about 800 amino acids, about 400 amino acids to about750 amino acids, about 400 amino acids to about 700 amino acids, about400 amino acids to about 650 amino acids, about 400 amino acids to about600 amino acids, about 400 amino acids to about 550 amino acids, about400 amino acids to about 500 amino acids, about 400 amino acids to about480 amino acids, about 400 amino acids to about 460 amino acids, about400 amino acids to about 440 amino acids, about 400 amino acids to about420 amino acids, about 420 amino acids to about 3000 amino acids, about420 amino acids to about 2500 amino acids, about 420 amino acids toabout 2000 amino acids, about 420 amino acids to about 1500 amino acids,about 420 amino acids to about 1000 amino acids, about 420 amino acidsto about 950 amino acids, about 420 amino acids to about 900 aminoacids, about 420 amino acids to about 850 amino acids, about 420 aminoacids to about 800 amino acids, about 420 amino acids to about 750 aminoacids, about 420 amino acids to about 700 amino acids, about 420 aminoacids to about 650 amino acids, about 420 amino acids to about 600 aminoacids, about 420 amino acids to about 550 amino acids, about 420 aminoacids to about 500 amino acids, about 420 amino acids to about 480 aminoacids, about 420 amino acids to about 460 amino acids, about 420 aminoacids to about 440 amino acids, about 440 amino acids to about 3000amino acids, about 440 amino acids to about 2500 amino acids, about 440amino acids to about 2000 amino acids, about 440 amino acids to about1500 amino acids, about 440 amino acids to about 1000 amino acids, about440 amino acids to about 950 amino acids, about 440 amino acids to about900 amino acids, about 440 amino acids to about 850 amino acids, about440 amino acids to about 800 amino acids, about 440 amino acids to about750 amino acids, about 440 amino acids to about 700 amino acids, about440 amino acids to about 650 amino acids, about 440 amino acids to about600 amino acids, about 440 amino acids to about 550 amino acids, about440 amino acids to about 500 amino acids, about 440 amino acids to about480 amino acids, about 440 amino acids to about 460 amino acids, about460 amino acids to about 3000 amino acids, about 460 amino acids toabout 2500 amino acids, about 460 amino acids to about 2000 amino acids,about 460 amino acids to about 1500 amino acids, about 460 amino acidsto about 1000 amino acids, about 460 amino acids to about 950 aminoacids, about 460 amino acids to about 900 amino acids, about 460 aminoacids to about 850 amino acids, about 460 amino acids to about 800 aminoacids, about 460 amino acids to about 750 amino acids, about 460 aminoacids to about 700 amino acids, about 460 amino acids to about 650 aminoacids, about 460 amino acids to about 600 amino acids, about 460 aminoacids to about 550 amino acids, about 460 amino acids to about 500 aminoacids, about 460 amino acids to about 480 amino acids, about 480 aminoacids to about 3000 amino acids, about 480 amino acids to about 2500amino acids, about 480 amino acids to about 2000 amino acids, about 480amino acids to about 1500 amino acids, about 480 amino acids to about1000 amino acids, about 480 amino acids to about 950 amino acids, about480 amino acids to about 900 amino acids, about 480 amino acids to about850 amino acids, about 480 amino acids to about 800 amino acids, about480 amino acids to about 750 amino acids, about 480 amino acids to about700 amino acids, about 480 amino acids to about 650 amino acids, about480 amino acids to about 600 amino acids, about 480 amino acids to about550 amino acids, about 480 amino acids to about 500 amino acids, about500 amino acids to about 3000 amino acids, about 500 amino acids toabout 2500 amino acids, about 500 amino acids to about 2000 amino acids,about 500 amino acids to about 1500 amino acids, about 500 amino acidsto about 1000 amino acids, about 500 amino acids to about 950 aminoacids, about 500 amino acids to about 900 amino acids, about 500 aminoacids to about 850 amino acids, about 500 amino acids to about 800 aminoacids, about 500 amino acids to about 750 amino acids, about 500 aminoacids to about 700 amino acids, about 500 amino acids to about 650 aminoacids, about 500 amino acids to about 600 amino acids, about 500 aminoacids to about 550 amino acids, about 550 amino acids to about 3000amino acids, about 550 amino acids to about 2500 amino acids, about 550amino acids to about 2000 amino acids, about 550 amino acids to about1500 amino acids, about 550 amino acids to about 1000 amino acids, about550 amino acids to about 950 amino acids, about 550 amino acids to about900 amino acids, about 550 amino acids to about 850 amino acids, about550 amino acids to about 800 amino acids, about 550 amino acids to about750 amino acids, about 550 amino acids to about 700 amino acids, about550 amino acids to about 650 amino acids, about 550 amino acids to about600 amino acids, about 600 amino acids to about 3000 amino acids, about600 amino acids to about 2500 amino acids, about 600 amino acids toabout 2000 amino acids, about 600 amino acids to about 1500 amino acids,about 600 amino acids to about 1000 amino acids, about 600 amino acidsto about 950 amino acids, about 600 amino acids to about 900 aminoacids, about 600 amino acids to about 850 amino acids, about 600 aminoacids to about 800 amino acids, about 600 amino acids to about 750 aminoacids, about 600 amino acids to about 700 amino acids, about 600 aminoacids to about 650 amino acids, about 650 amino acids to about 3000amino acids, about 650 amino acids to about 2500 amino acids, about 650amino acids to about 2000 amino acids, about 650 amino acids to about1500 amino acids, about 650 amino acids to about 1000 amino acids, about650 amino acids to about 950 amino acids, about 650 amino acids to about900 amino acids, about 650 amino acids to about 850 amino acids, about650 amino acids to about 800 amino acids, about 650 amino acids to about750 amino acids, about 650 amino acids to about 700 amino acids, about700 amino acids to about 3000 amino acids, about 700 amino acids toabout 2500 amino acids, about 700 amino acids to about 2000 amino acids,about 700 amino acids to about 1500 amino acids, about 700 amino acidsto about 1000 amino acids, about 700 amino acids to about 950 aminoacids, about 700 amino acids to about 900 amino acids, about 700 aminoacids to about 850 amino acids, about 700 amino acids to about 800 aminoacids, about 700 amino acids to about 750 amino acids, about 750 aminoacids to about 3000 amino acids, about 750 amino acids to about 2500amino acids, about 750 amino acids to about 2000 amino acids, about 750amino acids to about 1500 amino acids, about 750 amino acids to about1000 amino acids, about 750 amino acids to about 950 amino acids, about750 amino acids to about 900 amino acids, about 750 amino acids to about850 amino acids, about 750 amino acids to about 800 amino acids, about800 amino acids to about 3000 amino acids, about 800 amino acids toabout 2500 amino acids, about 800 amino acids to about 2000 amino acids,about 800 amino acids to about 1500 amino acids, about 800 amino acidsto about 1000 amino acids, about 800 amino acids to about 950 aminoacids, about 800 amino acids to about 900 amino acids, about 800 aminoacids to about 850 amino acids, about 850 amino acids to about 3000amino acids, about 850 amino acids to about 2500 amino acids, about 850amino acids to about 2000 amino acids, about 850 amino acids to about1500 amino acids, about 850 amino acids to about 1000 amino acids, about850 amino acids to about 950 amino acids, about 850 amino acids to about900 amino acids, about 900 amino acids to about 3000 amino acids, about900 amino acids to about 2500 amino acids, about 900 amino acids toabout 2000 amino acids, about 900 amino acids to about 1500 amino acids,about 900 amino acids to about 1000 amino acids, about 900 amino acidsto about 950 amino acids, about 950 amino acids to about 3000 aminoacids, about 950 amino acids to about 2500 amino acids, about 950 aminoacids to about 2000 amino acids, about 950 amino acids to about 1500amino acids, about 950 amino acids to about 1000 amino acids, about 1000amino acids to about 3000 amino acids, about 1000 amino acids to about2500 amino acids, about 1000 amino acids to about 2000 amino acids,about 1000 amino acids to about 1500 amino acids, about 1500 amino acidsto about 3000 amino acids, about 1500 amino acids to about 2500 aminoacids, about 1500 amino acids to about 2000 amino acids, about 2000amino acids to about 3000 amino acids, about 2000 amino acids to about2500 amino acids, or about 2500 amino acids to about 3000 amino acids.Diagrams of an exemplary single-chain chimeric polypeptide providedherein are depicted in FIG. 1 .

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain (e.g., any of theexemplary target-binding domains described herein or known in the art)and the soluble tissue factor domain (e.g., any of the exemplary solubletissue factor domains described herein) directly abut each other. Insome embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the single-chain chimeric polypeptide furthercomprises a linker sequence (e.g., any of the exemplary linker sequencesdescribed herein or known in the art) between the first target-bindingdomain (e.g., any of the exemplary target-binding domains describedherein or known in the art) and the soluble tissue factor domain (e.g.,any of the exemplary soluble tissue factor domains described herein). Insome embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the soluble tissue factor domain (e.g., any of theexemplary soluble tissue factor domains described herein) and the secondtarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art) directly abut each other. In someembodiments of any of the single-chain chimeric polypeptides describedherein, the single-chain chimeric polypeptide further comprises a linkersequence (e.g., any of the exemplary linker sequences described hereinor known in the art) between the soluble tissue factor domain (e.g., anyof the exemplary soluble tissue factor domains described herein) and thesecond target-binding domain (e.g., any of the exemplary target-bindingdomains described herein or known in the art).

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain (e.g., any of theexemplary target-binding domains described herein or known in the art)and the second target-binding domain (e.g., any of the exemplarytarget-binding domains described herein or known in the art) directlyabut each other. In some embodiments of any of the single-chain chimericpolypeptides described herein, the single-chain chimeric polypeptidefurther comprises a linker sequence (e.g., any of the exemplary linkersequences described herein or known in the art) between the firsttarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art) and the second target-bindingdomain (e.g., any of the exemplary target-binding domains describedherein or known in the art). In some embodiments of any of thesingle-chain chimeric polypeptides described herein, the secondtarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art) and the soluble tissue factordomain (e.g., any of the exemplary soluble tissue factor domainsdescribed herein) directly abut each other. In some embodiments of anyof the single-chain chimeric polypeptides described herein, thesingle-chain chimeric polypeptide further comprises a linker sequence(e.g., any of the exemplary linker sequences described herein or knownin the art) between the second target-binding domain (e.g., any of theexemplary target-binding domains described herein or known in the art)and the soluble tissue factor domain (e.g., any of the exemplary solubletissue factor domains described herein or known in the art).

In some embodiments, a single-chain chimeric polypeptide can include asequence that is at least 70% identical (e.g., at least 75% identical,at least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, at least 99% identical, or 100% identical) to

(SEQ ID NO: 1) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDATSKLSGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR.

In some embodiments, a single-chain chimeric polypeptide is encoded by anucleic acid that includes a sequence that is at least 70% identical(e.g., at least 75% identical, at least 80% identical, at least 85%identical, at least 90% identical, at least 95% identical, at least 99%identical, or 100% identical) to

(SEQ ID NO: 2) CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG.

In some embodiments, a single-chain chimeric polypeptide can include asequence that is at least 70% identical (e.g., at least 75% identical,at least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, at least 99% identical, or 100% identical) to

(SEQ ID NO: 3) MKWVTFISLLFLFSSAYSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR.

In some embodiments, a single-chain chimeric polypeptide is encoded by anucleic acid that includes a sequence that is at least 70% identical(e.g., at least 75% identical, at least 80% identical, at least 85%identical, at least 90% identical, at least 95% identical, at least 99%identical, or 100% identical) to

(SEQ ID NO: 4) ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCTATTCCCAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG.

In some embodiments, a single-chain chimeric polypeptide can include asequence that is at least 70% identical (e.g., at least 75% identical,at least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, at least 99% identical, or 100% identical) to

(SEQ ID NO: 5) VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKRSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYW GQGTTLTVSS.

In some embodiments, a single-chain chimeric polypeptide is encoded by anucleic acid that includes a sequence that is at least 70% identical(e.g., at least 75% identical, at least 80% identical, at least 85%identical, at least 90% identical, at least 95% identical, at least 99%identical, or 100% identical) to

(SEQ ID NO: 6) GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGCCGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGCGACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAGACCAAGCGGAGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACCGACGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCCTACCCCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGAGAGTTTCGTGAGCAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGTGGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGGAGGTAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC.

In some embodiments, a single-chain chimeric polypeptide can include asequence that is at least 70% identical (e.g., at least 75% identical,at least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, at least 99% identical, or 100% identical) to

(SEQ ID NO: 7) MKWVTFISLLFLFSSAYSVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKRSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC LDYWGQGTTLTVSS.

In some embodiments, a single-chain chimeric polypeptide is encoded by anucleic acid that includes a sequence that is at least 70% identical(e.g., at least 75% identical, at least 80% identical, at least 85%identical, at least 90% identical, at least 95% identical, at least 99%identical, or 100% identical) to

(SEQ ID NO: 8) ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTTATTTAGCAGCGCCTACAGCGTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGCCGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGCGACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAGACCAAGCGGAGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACCGACGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCCTACCCCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGAGAGTTTCGTGAGCAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGTGGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGGAGGTAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC.

Non-limiting aspects of these chimeric polypeptides, nucleic acids,vectors, cells, and methods are described below, and can be used in anycombination without limitation. Additional aspects of these chimericpolypeptides, nucleic acids, vectors, cells, and methods are known inthe art.

Tissue Factor

Human tissue factor is a 263 amino-acid transmembrane protein containingthree domains: (1) a 219-amino acid N-terminal extracellular domain(residues 1-219); (2) a 22-amino acid transmembrane domain (residues220-242); and (3) a 21-amino acid cytoplasmic C-terminal tail (residues242-263) ((UniProtKB Identifier Number: P13726). The cytoplasmic tailcontains two phosphorylation sites at Ser253 and Ser258, and oneS-palmitoylation site at Cys245. Deletion or mutation of the cytoplasmicdomain was not found to affect tissue factor coagulation activity.Tissue factor has one S-palmitoylation site in the intracellular domainof the protein at Cys245. The Cys245 is located at the amino acidterminus of the intracellular domain and close to the membrane surface.The tissue factor transmembrane domain is composed of a single-spanningα-helix.

The extracellular domain of tissue factor, composed of two fibronectintype III domains, is connected to the transmembrane domain through asix-amino acid linker. This linker provides conformational flexibilityto decouple the tissue factor extracellular domain from itstransmembrane and cytoplasmic domains. Each tissue factor fibronectintype III module is composed of two overlapping β sheets with the topsheet domain containing three antiparallel β-strands and the bottomsheet containing four β-strands. The β-strands are connected by 0-loopsbetween strand βA and βB, βC and βD, and βE and βF, all of which areconserved in conformation in the two modules. There are three shortα-helix segments connecting the β-strands. A unique feature of tissuefactor is a 17-amino acid β-hairpin between strand β10 and strand β11,which is not a common element of the fibronectin superfamily. TheN-terminal domain also contains a 12 amino acid loop between β6F and β7Gthat is not present in the C-terminal domain and is unique to tissuefactor. Such a fibronectin type III domain structure is a feature of theimmunoglobulin-like family of protein folds and is conserved among awide variety of extracellular proteins.

The zymogen FVII is rapidly converted to FVIIa by limited proteolysisonce it binds to tissue to form the active tissue factor-FVIIa complex.The FVIIa, which circulates as an enzyme at a concentration ofapproximately 0.1 nM (1% of plasma FVII), can also bind directly totissue factor. The allosteric interaction between tissue factor andFVIIa on the tissue factor-FVIIa complex greatly increases the enzymaticactivity of FVIIa: an approximate 20- to 100-fold increase in the rateof hydrolysis of small, chromogenic peptidyl substrates, and nearly amillion-fold increase in the rate of activation of the naturalmacromolecular substrates FIX and FX. In concert with allostericactivation of the active site of FVIIa upon binding to tissue factor,the formation of tissue factor-FVIIa complex on phospholipid bilayer(i.e., upon exposure of phosphatidyl-L-serine on membrane surfaces)increases the rate of FIX or FX activation, in a Ca²⁺-dependent manner,an additional 1,000-fold. The roughly million-fold overall increase inFX activation by tissue factor-FVIIa-phospholipid complex relative tofree FVIIa is a critical regulatory point for the coagulation cascade.

FVII is a ˜50 kDa, single-chain polypeptide consisting of 406 amino acidresidues, with an N-terminal γ-carboxyglutamate-rich (GLA) domain, twoepidermal growth factor-like domains (EGF1 and EFG2), and a C-terminalserine protease domain. FVII is activated to FVIIa by a specificproteolytic cleavage of the Ile-¹⁵⁴-Arg¹⁵² bond in the short linkerregion between the EGF2 and the protease domain. This cleavage resultsin the light and heavy chains being held together by a single disulfidebond of Cys¹³⁵ and Cys²⁶². FVIIa binds phospholipid membrane in aCa²⁺-dependent manner through its N-terminal GLA-domain. ImmediatelyC-terminal to the GLA domain is an aromatic stack and two EGF domains.The aromatic stack connects the GLA to EGF1 domain which binds a singleCa²⁺ ion. Occupancy of this Ca²⁺-binding site increases FVIIa amidolyticactivity and tissue factor association. The catalytic triad consist ofHis¹⁹³, Asp²⁴², and Ser³⁴⁴, and binding of a single Ca²⁺ ion within theFVIIa protease domain is critical for its catalytic activity.Proteolytic activation of FVII to FVIIa frees the newly formed aminoterminus at Ile¹⁵³ to fold back and be inserted into the activationpocket forming a salt bridge with the carboxylate of Asp³⁴³ to generatethe oxyanion hole. Formation of this salt bridge is critical for FVIIaactivity. However, oxyanion hole formation does not occur in free FVIIaupon proteolytic activation. As a result, FVIIa circulates in azymogen-like state that is poorly recognized by plasma proteaseinhibitors, allowing it to circulate with a half-life of approximately90 minutes.

Tissue factor-mediated positioning of the FVIIa active site above themembrane surface is important for FVIIa towards cognate substrates. FreeFVIIa adopts a stable, extended structure when bound to the membranewith its active site positioned ˜80 Å above the membrane surface. UponFVIIa binding to tissue factor, the FVa active site is repositioned ˜6 Åcloser to the membrane. This modulation may aid in a proper alignment ofthe FVIIa catalytic triad with the target substrate cleavage site. UsingGLA-domainless FVIIa, it has been shown that the active site was stillpositioned a similar distance above the membrane, demonstrating thattissue factor is able to fully support FVIIa active site positioningeven in the absence of FVIIa-membrane interaction. Additional datashowed that tissue factor supported full FVIIa proteolytic activity aslong as the tissue factor extracellular domain was tethered in some wayto the membrane surface. However, raising the active site of FVIIagreater than 80 Å above the membrane surface greatly reduced the abilityof the tissue factor-FVIIa complex to activate FX but did not diminishtissue factor-FVIIa amidolytic activity.

Alanine scanning mutagenesis has been used to assess the role ofspecific amino acid side chains in the tissue factor extracellulardomain for interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., J Biol Chem 269(30): 19399-19403,1994). Alanine substitution identified a limited number of residuepositions at which alanine replacements cause 5- to 10-fold loweraffinity for FVIIa binding. Most of these residue side chains were foundto be well-exposed to solvent in the crystal structure, concordant withmacromolecular ligand interaction. The FVIIa ligand-binding site islocated over an extensive region at the boundary between the twomodules. In the C-module, residues Arg¹³⁵ and Phe¹⁴⁰ located on theprotruding B-C loop provide an independent contact with FVIIa. Leu¹³³ islocated at the base of the fingerlike structure and packed into thecleft between the two modules. This provides continuity to a majorcluster of important binding residues consisting of Lys²⁰, Thr⁶⁰, Asp⁵⁸,and Ile²². Thr⁶⁰ is only partially solvent-exposed and may play a localstructural role rather than making a significant contact with ligand.The binding site extends onto the concave side of the intermodule angleinvolving Glu²⁴ and Gln¹¹⁰, and potentially the more distant residueVal²⁰⁷. The binding region extends from Asp⁵⁸ onto a convex surface areaformed by Lys⁴⁸, Lys⁴⁶, Gln³⁷, Asp⁴⁴, and Trp⁴⁵. Trp⁴⁵ and Asp⁴⁴ do notinteract independently with FVIIa, indicating that the mutational effectat the Trp⁴⁵ position may reflect a structural importance of this sidechain for the local packing of the adjacent Asp⁴⁴ and Gln³⁷ side chain.The interactive area further includes two surface-exposed aromaticresidues, Phe⁷⁶ and Tyr⁷⁸, which form part of the hydrophobic cluster inthe N-module.

The known physiologic substrates of tissue factor-FVIIa are FVII, FIX,and FX and certain proteinase-activated receptors. Mutational analysishas identified a number of residues that, when mutated, support fullFVIIa amidolytic activity towards small peptidyl substrates but aredeficient in their ability to support macromolecular substrate (i.e.,FVII, FIX, and FX) activation (Ruf et al., J Biol Chem 267(31):22206-22210, 1992; Ruf et al., J Biol Chem 267(9): 6375-6381, 1992;Huang et al., J Biol Chem 271(36): 21752-21757, 1996; Kirchhofer et al.,Biochemistry 39(25): 7380-7387, 2000). The tissue factor loop region atresidues 159-165, and residues in or adjacent to this flexible loop havebeen shown to be critical for the proteolytic activity of the tissuefactor-FVIIa complex. This defines the proposed substrate-bindingexosite region of tissue factor that is quite distant from the FVIIaactive site. A substitution of the glycine residue by a marginallybulkier residue alanine, significantly impairs tissue factor-FVIIaproteolytic activity. This suggests that the flexibility afforded byglycine is critical for the loop of residues 159-165 for tissue factormacromolecular substrate recognition.

The residues Lys¹⁶⁵ and Lys¹⁶⁶ have also been demonstrated to beimportant for substrate recognition and binding. Mutation of either ofthese residues to alanine results in a significant decrease in thetissue factor co-factor function. Lys¹⁶⁵ and Lys¹⁶⁶ face away from eachother, with Lys¹⁶⁵ pointing towards FVIIa in most tissue factor-FVIIastructures, and Lys¹⁶⁶ pointing into the substrate binding exositeregion in the crystal structure. Putative salt bridge formation betweenLys¹⁶⁵ of and Gla³⁵ of FVIIa would support the notion that tissue factorinteraction with the GLA domain of FVIIa modulates substraterecognition. These results suggest that the C-terminal portion of thetissue factor ectodomain directly interacts with the GLA-domain, thepossible adjacent EGF1 domains, of FIX and FX, and that the presence ofthe FVIIa GLA-domain may modulate these interactions either directly orindirectly.

Soluble Tissue Factor Domain

In some embodiments of any of the single-chain chimeric polypeptides,compositions, or methods described herein, the soluble tissue factordomain can be a wildtype tissue factor polypeptide lacking the signalsequence, the transmembrane domain, and the intracellular domain. Insome examples, the soluble tissue factor domain can be a tissue factormutant, wherein a wildtype tissue factor polypeptide lacking the signalsequence, the transmembrane domain, and the intracellular domain, andhas been further modified at selected amino acids. In some examples, thesoluble tissue factor domain can be a soluble human tissue factordomain. In some examples, the soluble tissue factor domain can be asoluble mouse tissue factor domain. In some examples, the soluble tissuefactor domain can be a soluble rat tissue factor domain. Non-limitingexamples of soluble human tissue factor domains, a mouse soluble tissuefactor domain, a rat soluble tissue factor domain, and mutant solubletissue factor domains are shown below.

Exemplary Soluble Human Tissue Factor Domain (SEQ ID NO: 9)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFREExemplary Nucleic Acid Encoding Soluble Human Tissue Factor Domain(SEQ ID NO: 10) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAGExemplary Soluble Mouse Tissue Factor Domain (SEQ ID NO: 11)agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnapkflpyrdtnlgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstveteqwksflge Exemplary Soluble Rat Tissue Factor Domain(SEQ ID NO: 12) Agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtkigqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge Exemplary Mutant Soluble Human Tissue FactorDomain (SEQ ID NO: 96)SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFREExemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO: 97)SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE

In some embodiments, a soluble tissue factor domain can include asequence that is at least 70% identical, at least 72% identical, atleast 74% identical, at least 76% identical, at least 78% identical, atleast 80% identical, at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical to SEQ IDNO: 9, 11, 12, 96, or 97. In some embodiments, a soluble tissue factordomain can include a sequence of SEQ ID NO: 9, 11, 12, 96, or 97 withone to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20) amino acids removed from its N-terminusand/or one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids removed from itsC-terminus.

As can be appreciated in the art, one skilled in the art wouldunderstand that mutation of amino acids that are conserved betweendifferent mammalian species is more likely to decrease the activityand/or structural stability of the protein, while mutation of aminoacids that are not conserved between different mammalian species is lesslikely to decrease the activity and/or structural stability of theprotein.

In some examples of any of the single-chain chimeric polypeptidesdescribed herein, the soluble tissue factor domain is not capable ofbinding to Factor VIIa. In some examples of any of the single-chainchimeric polypeptides described herein, the soluble tissue factor domaindoes not convert inactive Factor X into Factor Xa. In some embodimentsof any of the single-chain chimeric polypeptides described herein, thesingle-chain chimeric polypeptide does not stimulate blood coagulationin a mammal.

In some examples, the soluble tissue factor domain can be a solublehuman tissue factor domain. In some embodiments, the soluble tissuefactor domain can be a soluble mouse tissue factor domain. In someembodiments, the soluble tissue factor domain can be a soluble rattissue factor domain.

In some examples, the soluble tissue factor domain does not include oneor more (e.g., two, three, four, five, six or seven) of: a lysine at anamino acid position that corresponds to amino acid position 20 of maturewildtype human tissue factor protein; an isoleucine at an amino acidposition that corresponds to amino acid position 22 of mature wildtypehuman tissue factor protein; a tryptophan at an amino acid position thatcorresponds to amino acid position 45 of mature wildtype human tissuefactor protein; an aspartic acid at an amino acid position thatcorresponds to amino acid position 58 of mature wildtype human tissuefactor protein; a tyrosine at an amino acid position that corresponds toamino acid position 94 of mature wildtype human tissue factor protein;an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and aphenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein. In someembodiments, the mutant soluble tissue factor possesses the amino acidsequence of SEQ ID NO: 96 or SEQ ID NO: 97.

In some examples, the soluble tissue factor domain can be encoded by anucleic acid including a sequence that is at least 70% identical, atleast 72% identical, at least 74% identical, at least 76% identical, atleast 78% identical, at least 80% identical, at least 82% identical, atleast 84% identical, at least 86% identical, at least 88% identical, atleast 90% identical, at least 92% identical, at least 94% identical, atleast 96% identical, at least 98% identical, at least 99% identical, or100% identical to SEQ ID NO: 10.

In some embodiments, the soluble tissue factor domain can have a totallength of about 20 amino acids to about 220 amino acids, about 20 aminoacids to about 215 amino acids, about 20 amino acids to about 210 aminoacids, about 20 amino acids to about 205 amino acids, about 20 aminoacids to about 200 amino acids, about 20 amino acids to about 195 aminoacids, about 20 amino acids to about 190 amino acids, about 20 aminoacids to about 185 amino acids, about 20 amino acids to about 180 aminoacids, about 20 amino acids to about 175 amino acids, about 20 aminoacids to about 170 amino acids, about 20 amino acids to about 165 aminoacids, about 20 amino acids to about 160 amino acids, about 20 aminoacids to about 155 amino acids, about 20 amino acids to about 150 aminoacids, about 20 amino acids to about 145 amino acids, about 20 aminoacids to about 140 amino acids, about 20 amino acids to about 135 aminoacids, about 20 amino acids to about 130 amino acids, about 20 aminoacids to about 125 amino acids, about 20 amino acids to about 120 aminoacids, about 20 amino acids to about 115 amino acids, about 20 aminoacids to about 110 amino acids, about 20 amino acids to about 105 aminoacids, about 20 amino acids to about 100 amino acids, about 20 aminoacids to about 95 amino acids, about 20 amino acids to about 90 aminoacids, about 20 amino acids to about 85 amino acids, about 20 aminoacids to about 80 amino acids, about 20 amino acids to about 75 aminoacids, about 20 amino acids to about 70 amino acids, about 20 aminoacids to about 60 amino acids, about 20 amino acids to about 50 aminoacids, about 20 amino acids to about 40 amino acids, about 20 aminoacids to about 30 amino acids, about 30 amino acids to about 220 aminoacids, about 30 amino acids to about 215 amino acids, about 30 aminoacids to about 210 amino acids, about 30 amino acids to about 205 aminoacids, about 30 amino acids to about 200 amino acids, about 30 aminoacids to about 195 amino acids, about 30 amino acids to about 190 aminoacids, about 30 amino acids to about 185 amino acids, about 30 aminoacids to about 180 amino acids, about 30 amino acids to about 175 aminoacids, about 30 amino acids to about 170 amino acids, about 30 aminoacids to about 165 amino acids, about 30 amino acids to about 160 aminoacids, about 30 amino acids to about 155 amino acids, about 30 aminoacids to about 150 amino acids, about 30 amino acids to about 145 aminoacids, about 30 amino acids to about 140 amino acids, about 30 aminoacids to about 135 amino acids, about 30 amino acids to about 130 aminoacids, about 30 amino acids to about 125 amino acids, about 30 aminoacids to about 120 amino acids, about 30 amino acids to about 115 aminoacids, about 30 amino acids to about 110 amino acids, about 30 aminoacids to about 105 amino acids, about 30 amino acids to about 100 aminoacids, about 30 amino acids to about 95 amino acids, about 30 aminoacids to about 90 amino acids, about 30 amino acids to about 85 aminoacids, about 30 amino acids to about 80 amino acids, about 30 aminoacids to about 75 amino acids, about 30 amino acids to about 70 aminoacids, about 30 amino acids to about 60 amino acids, about 30 aminoacids to about 50 amino acids, about 30 amino acids to about 40 aminoacids, about 40 amino acids to about 220 amino acids, about 40 aminoacids to about 215 amino acids, about 40 amino acids to about 210 aminoacids, about 40 amino acids to about 205 amino acids, about 40 aminoacids to about 200 amino acids, about 40 amino acids to about 195 aminoacids, about 40 amino acids to about 190 amino acids, about 40 aminoacids to about 185 amino acids, about 40 amino acids to about 180 aminoacids, about 40 amino acids to about 175 amino acids, about 40 aminoacids to about 170 amino acids, about 40 amino acids to about 165 aminoacids, about 40 amino acids to about 160 amino acids, about 40 aminoacids to about 155 amino acids, about 40 amino acids to about 150 aminoacids, about 40 amino acids to about 145 amino acids, about 40 aminoacids to about 140 amino acids, about 40 amino acids to about 135 aminoacids, about 40 amino acids to about 130 amino acids, about 40 aminoacids to about 125 amino acids, about 40 amino acids to about 120 aminoacids, about 40 amino acids to about 115 amino acids, about 40 aminoacids to about 110 amino acids, about 40 amino acids to about 105 aminoacids, about 40 amino acids to about 100 amino acids, about 40 aminoacids to about 95 amino acids, about 40 amino acids to about 90 aminoacids, about 40 amino acids to about 85 amino acids, about 40 aminoacids to about 80 amino acids, about 40 amino acids to about 75 aminoacids, about 40 amino acids to about 70 amino acids, about 40 aminoacids to about 60 amino acids, about 40 amino acids to about 50 aminoacids, about 50 amino acids to about 220 amino acids, about 50 aminoacids to about 215 amino acids, about 50 amino acids to about 210 aminoacids, about 50 amino acids to about 205 amino acids, about 50 aminoacids to about 200 amino acids, about 50 amino acids to about 195 aminoacids, about 50 amino acids to about 190 amino acids, about 50 aminoacids to about 185 amino acids, about 50 amino acids to about 180 aminoacids, about 50 amino acids to about 175 amino acids, about 50 aminoacids to about 170 amino acids, about 50 amino acids to about 165 aminoacids, about 50 amino acids to about 160 amino acids, about 50 aminoacids to about 155 amino acids, about 50 amino acids to about 150 aminoacids, about 50 amino acids to about 145 amino acids, about 50 aminoacids to about 140 amino acids, about 50 amino acids to about 135 aminoacids, about 50 amino acids to about 130 amino acids, about 50 aminoacids to about 125 amino acids, about 50 amino acids to about 120 aminoacids, about 50 amino acids to about 115 amino acids, about 50 aminoacids to about 110 amino acids, about 50 amino acids to about 105 aminoacids, about 50 amino acids to about 100 amino acids, about 50 aminoacids to about 95 amino acids, about 50 amino acids to about 90 aminoacids, about 50 amino acids to about 85 amino acids, about 50 aminoacids to about 80 amino acids, about 50 amino acids to about 75 aminoacids, about 50 amino acids to about 70 amino acids, about 50 aminoacids to about 60 amino acids, about 60 amino acids to about 220 aminoacids, about 60 amino acids to about 215 amino acids, about 60 aminoacids to about 210 amino acids, about 60 amino acids to about 205 aminoacids, about 60 amino acids to about 200 amino acids, about 60 aminoacids to about 195 amino acids, about 60 amino acids to about 190 aminoacids, about 60 amino acids to about 185 amino acids, about 60 aminoacids to about 180 amino acids, about 60 amino acids to about 175 aminoacids, about 60 amino acids to about 170 amino acids, about 60 aminoacids to about 165 amino acids, about 60 amino acids to about 160 aminoacids, about 60 amino acids to about 155 amino acids, about 60 aminoacids to about 150 amino acids, about 60 amino acids to about 145 aminoacids, about 60 amino acids to about 140 amino acids, about 60 aminoacids to about 135 amino acids, about 60 amino acids to about 130 aminoacids, about 60 amino acids to about 125 amino acids, about 60 aminoacids to about 120 amino acids, about 60 amino acids to about 115 aminoacids, about 60 amino acids to about 110 amino acids, about 60 aminoacids to about 105 amino acids, about 60 amino acids to about 100 aminoacids, about 60 amino acids to about 95 amino acids, about 60 aminoacids to about 90 amino acids, about 60 amino acids to about 85 aminoacids, about 60 amino acids to about 80 amino acids, about 60 aminoacids to about 75 amino acids, about 60 amino acids to about 70 aminoacids, about 70 amino acids to about 220 amino acids, about 70 aminoacids to about 215 amino acids, about 70 amino acids to about 210 aminoacids, about 70 amino acids to about 205 amino acids, about 70 aminoacids to about 200 amino acids, about 70 amino acids to about 195 aminoacids, about 70 amino acids to about 190 amino acids, about 70 aminoacids to about 185 amino acids, about 70 amino acids to about 180 aminoacids, about 70 amino acids to about 175 amino acids, about 70 aminoacids to about 170 amino acids, about 70 amino acids to about 165 aminoacids, about 70 amino acids to about 160 amino acids, about 70 aminoacids to about 155 amino acids, about 70 amino acids to about 150 aminoacids, about 70 amino acids to about 145 amino acids, about 70 aminoacids to about 140 amino acids, about 70 amino acids to about 135 aminoacids, about 70 amino acids to about 130 amino acids, about 70 aminoacids to about 125 amino acids, about 70 amino acids to about 120 aminoacids, about 70 amino acids to about 115 amino acids, about 70 aminoacids to about 110 amino acids, about 70 amino acids to about 105 aminoacids, about 70 amino acids to about 100 amino acids, about 70 aminoacids to about 95 amino acids, about 70 amino acids to about 90 aminoacids, about 70 amino acids to about 85 amino acids, about 70 aminoacids to about 80 amino acids, about 80 amino acids to about 220 aminoacids, about 80 amino acids to about 215 amino acids, about 80 aminoacids to about 210 amino acids, about 80 amino acids to about 205 aminoacids, about 80 amino acids to about 200 amino acids, about 80 aminoacids to about 195 amino acids, about 80 amino acids to about 190 aminoacids, about 80 amino acids to about 185 amino acids, about 80 aminoacids to about 180 amino acids, about 80 amino acids to about 175 aminoacids, about 80 amino acids to about 170 amino acids, about 80 aminoacids to about 165 amino acids, about 80 amino acids to about 160 aminoacids, about 80 amino acids to about 155 amino acids, about 80 aminoacids to about 150 amino acids, about 80 amino acids to about 145 aminoacids, about 80 amino acids to about 140 amino acids, about 80 aminoacids to about 135 amino acids, about 80 amino acids to about 130 aminoacids, about 80 amino acids to about 125 amino acids, about 80 aminoacids to about 120 amino acids, about 80 amino acids to about 115 aminoacids, about 80 amino acids to about 110 amino acids, about 80 aminoacids to about 105 amino acids, about 80 amino acids to about 100 aminoacids, about 80 amino acids to about 95 amino acids, about 80 aminoacids to about 90 amino acids, about 90 amino acids to about 220 aminoacids, about 90 amino acids to about 215 amino acids, about 90 aminoacids to about 210 amino acids, about 90 amino acids to about 205 aminoacids, about 90 amino acids to about 200 amino acids, about 90 aminoacids to about 195 amino acids, about 90 amino acids to about 190 aminoacids, about 90 amino acids to about 185 amino acids, about 90 aminoacids to about 180 amino acids, about 90 amino acids to about 175 aminoacids, about 90 amino acids to about 170 amino acids, about 90 aminoacids to about 165 amino acids, about 90 amino acids to about 160 aminoacids, about 90 amino acids to about 155 amino acids, about 90 aminoacids to about 150 amino acids, about 90 amino acids to about 145 aminoacids, about 90 amino acids to about 140 amino acids, about 90 aminoacids to about 135 amino acids, about 90 amino acids to about 130 aminoacids, about 90 amino acids to about 125 amino acids, about 90 aminoacids to about 120 amino acids, about 90 amino acids to about 115 aminoacids, about 90 amino acids to about 110 amino acids, about 90 aminoacids to about 105 amino acids, about 90 amino acids to about 100 aminoacids, about 100 amino acids to about 220 amino acids, about 100 aminoacids to about 215 amino acids, about 100 amino acids to about 210 aminoacids, about 100 amino acids to about 205 amino acids, about 100 aminoacids to about 200 amino acids, about 100 amino acids to about 195 aminoacids, about 100 amino acids to about 190 amino acids, about 100 aminoacids to about 185 amino acids, about 100 amino acids to about 180 aminoacids, about 100 amino acids to about 175 amino acids, about 100 aminoacids to about 170 amino acids, about 100 amino acids to about 165 aminoacids, about 100 amino acids to about 160 amino acids, about 100 aminoacids to about 155 amino acids, about 100 amino acids to about 150 aminoacids, about 100 amino acids to about 145 amino acids, about 100 aminoacids to about 140 amino acids, about 100 amino acids to about 135 aminoacids, about 100 amino acids to about 130 amino acids, about 100 aminoacids to about 125 amino acids, about 100 amino acids to about 120 aminoacids, about 100 amino acids to about 115 amino acids, about 100 aminoacids to about 110 amino acids, about 110 amino acids to about 220 aminoacids, about 110 amino acids to about 215 amino acids, about 110 aminoacids to about 210 amino acids, about 110 amino acids to about 205 aminoacids, about 110 amino acids to about 200 amino acids, about 110 aminoacids to about 195 amino acids, about 110 amino acids to about 190 aminoacids, about 110 amino acids to about 185 amino acids, about 110 aminoacids to about 180 amino acids, about 110 amino acids to about 175 aminoacids, about 110 amino acids to about 170 amino acids, about 110 aminoacids to about 165 amino acids, about 110 amino acids to about 160 aminoacids, about 110 amino acids to about 155 amino acids, about 110 aminoacids to about 150 amino acids, about 110 amino acids to about 145 aminoacids, about 110 amino acids to about 140 amino acids, about 110 aminoacids to about 135 amino acids, about 110 amino acids to about 130 aminoacids, about 110 amino acids to about 125 amino acids, about 110 aminoacids to about 120 amino acids, about 110 amino acids to about 115 aminoacids, about 115 amino acids to about 220 amino acids, about 115 aminoacids to about 215 amino acids, about 115 amino acids to about 210 aminoacids, about 115 amino acids to about 205 amino acids, about 115 aminoacids to about 200 amino acids, about 115 amino acids to about 195 aminoacids, about 115 amino acids to about 190 amino acids, about 115 aminoacids to about 185 amino acids, about 115 amino acids to about 180 aminoacids, about 115 amino acids to about 175 amino acids, about 115 aminoacids to about 170 amino acids, about 115 amino acids to about 165 aminoacids, about 115 amino acids to about 160 amino acids, about 115 aminoacids to about 155 amino acids, about 115 amino acids to about 150 aminoacids, about 115 amino acids to about 145 amino acids, about 115 aminoacids to about 140 amino acids, about 115 amino acids to about 135 aminoacids, about 115 amino acids to about 130 amino acids, about 115 aminoacids to about 125 amino acids, about 115 amino acids to about 120 aminoacids, about 120 amino acids to about 220 amino acids, about 120 aminoacids to about 215 amino acids, about 120 amino acids to about 210 aminoacids, about 120 amino acids to about 205 amino acids, about 120 aminoacids to about 200 amino acids, about 120 amino acids to about 195 aminoacids, about 120 amino acids to about 190 amino acids, about 120 aminoacids to about 185 amino acids, about 120 amino acids to about 180 aminoacids, about 120 amino acids to about 175 amino acids, about 120 aminoacids to about 170 amino acids, about 120 amino acids to about 165 aminoacids, about 120 amino acids to about 160 amino acids, about 120 aminoacids to about 155 amino acids, about 120 amino acids to about 150 aminoacids, about 120 amino acids to about 145 amino acids, about 120 aminoacids to about 140 amino acids, about 120 amino acids to about 135 aminoacids, about 120 amino acids to about 130 amino acids, about 120 aminoacids to about 125 amino acids, about 125 amino acids to about 220 aminoacids, about 125 amino acids to about 215 amino acids, about 125 aminoacids to about 210 amino acids, about 125 amino acids to about 205 aminoacids, about 125 amino acids to about 200 amino acids, about 125 aminoacids to about 195 amino acids, about 125 amino acids to about 190 aminoacids, about 125 amino acids to about 185 amino acids, about 125 aminoacids to about 180 amino acids, about 125 amino acids to about 175 aminoacids, about 125 amino acids to about 170 amino acids, about 125 aminoacids to about 165 amino acids, about 125 amino acids to about 160 aminoacids, about 125 amino acids to about 155 amino acids, about 125 aminoacids to about 150 amino acids, about 125 amino acids to about 145 aminoacids, about 125 amino acids to about 140 amino acids, about 125 aminoacids to about 135 amino acids, about 125 amino acids to about 130 aminoacids, about 130 amino acids to about 220 amino acids, about 130 aminoacids to about 215 amino acids, about 130 amino acids to about 210 aminoacids, about 130 amino acids to about 205 amino acids, about 130 aminoacids to about 200 amino acids, about 130 amino acids to about 195 aminoacids, about 130 amino acids to about 190 amino acids, about 130 aminoacids to about 185 amino acids, about 130 amino acids to about 180 aminoacids, about 130 amino acids to about 175 amino acids, about 130 aminoacids to about 170 amino acids, about 130 amino acids to about 165 aminoacids, about 130 amino acids to about 160 amino acids, about 130 aminoacids to about 155 amino acids, about 130 amino acids to about 150 aminoacids, about 130 amino acids to about 145 amino acids, about 130 aminoacids to about 140 amino acids, about 130 amino acids to about 135 aminoacids, about 135 amino acids to about 220 amino acids, about 135 aminoacids to about 215 amino acids, about 135 amino acids to about 210 aminoacids, about 135 amino acids to about 205 amino acids, about 135 aminoacids to about 200 amino acids, about 135 amino acids to about 195 aminoacids, about 135 amino acids to about 190 amino acids, about 135 aminoacids to about 185 amino acids, about 135 amino acids to about 180 aminoacids, about 135 amino acids to about 175 amino acids, about 135 aminoacids to about 170 amino acids, about 135 amino acids to about 165 aminoacids, about 135 amino acids to about 160 amino acids, about 135 aminoacids to about 155 amino acids, about 135 amino acids to about 150 aminoacids, about 135 amino acids to about 145 amino acids, about 135 aminoacids to about 140 amino acids, about 140 amino acids to about 220 aminoacids, about 140 amino acids to about 215 amino acids, about 140 aminoacids to about 210 amino acids, about 140 amino acids to about 205 aminoacids, about 140 amino acids to about 200 amino acids, about 140 aminoacids to about 195 amino acids, about 140 amino acids to about 190 aminoacids, about 140 amino acids to about 185 amino acids, about 140 aminoacids to about 180 amino acids, about 140 amino acids to about 175 aminoacids, about 140 amino acids to about 170 amino acids, about 140 aminoacids to about 165 amino acids, about 140 amino acids to about 160 aminoacids, about 140 amino acids to about 155 amino acids, about 140 aminoacids to about 150 amino acids, about 140 amino acids to about 145 aminoacids, about 145 amino acids to about 220 amino acids, about 145 aminoacids to about 215 amino acids, about 145 amino acids to about 210 aminoacids, about 145 amino acids to about 205 amino acids, about 145 aminoacids to about 200 amino acids, about 145 amino acids to about 195 aminoacids, about 145 amino acids to about 190 amino acids, about 145 aminoacids to about 185 amino acids, about 145 amino acids to about 180 aminoacids, about 145 amino acids to about 175 amino acids, about 145 aminoacids to about 170 amino acids, about 145 amino acids to about 165 aminoacids, about 145 amino acids to about 160 amino acids, about 145 aminoacids to about 155 amino acids, about 145 amino acids to about 150 aminoacids, about 150 amino acids to about 220 amino acids, about 150 aminoacids to about 215 amino acids, about 150 amino acids to about 210 aminoacids, about 150 amino acids to about 205 amino acids, about 150 aminoacids to about 200 amino acids, about 150 amino acids to about 195 aminoacids, about 150 amino acids to about 190 amino acids, about 150 aminoacids to about 185 amino acids, about 150 amino acids to about 180 aminoacids, about 150 amino acids to about 175 amino acids, about 150 aminoacids to about 170 amino acids, about 150 amino acids to about 165 aminoacids, about 150 amino acids to about 160 amino acids, about 150 aminoacids to about 155 amino acids, about 155 amino acids to about 220 aminoacids, about 155 amino acids to about 215 amino acids, about 155 aminoacids to about 210 amino acids, about 155 amino acids to about 205 aminoacids, about 155 amino acids to about 200 amino acids, about 155 aminoacids to about 195 amino acids, about 155 amino acids to about 190 aminoacids, about 155 amino acids to about 185 amino acids, about 155 aminoacids to about 180 amino acids, about 155 amino acids to about 175 aminoacids, about 155 amino acids to about 170 amino acids, about 155 aminoacids to about 165 amino acids, about 155 amino acids to about 160 aminoacids, about 160 amino acids to about 220 amino acids, about 160 aminoacids to about 215 amino acids, about 160 amino acids to about 210 aminoacids, about 160 amino acids to about 205 amino acids, about 160 aminoacids to about 200 amino acids, about 160 amino acids to about 195 aminoacids, about 160 amino acids to about 190 amino acids, about 160 aminoacids to about 185 amino acids, about 160 amino acids to about 180 aminoacids, about 160 amino acids to about 175 amino acids, about 160 aminoacids to about 170 amino acids, about 160 amino acids to about 165 aminoacids, about 165 amino acids to about 220 amino acids, about 165 aminoacids to about 215 amino acids, about 165 amino acids to about 210 aminoacids, about 165 amino acids to about 205 amino acids, about 165 aminoacids to about 200 amino acids, about 165 amino acids to about 195 aminoacids, about 165 amino acids to about 190 amino acids, about 165 aminoacids to about 185 amino acids, about 165 amino acids to about 180 aminoacids, about 165 amino acids to about 175 amino acids, about 165 aminoacids to about 170 amino acids, about 170 amino acids to about 220 aminoacids, about 170 amino acids to about 215 amino acids, about 170 aminoacids to about 210 amino acids, about 170 amino acids to about 205 aminoacids, about 170 amino acids to about 200 amino acids, about 170 aminoacids to about 195 amino acids, about 170 amino acids to about 190 aminoacids, about 170 amino acids to about 185 amino acids, about 170 aminoacids to about 180 amino acids, about 170 amino acids to about 175 aminoacids, about 175 amino acids to about 220 amino acids, about 175 aminoacids to about 215 amino acids, about 175 amino acids to about 210 aminoacids, about 175 amino acids to about 205 amino acids, about 175 aminoacids to about 200 amino acids, about 175 amino acids to about 195 aminoacids, about 175 amino acids to about 190 amino acids, about 175 aminoacids to about 185 amino acids, about 175 amino acids to about 180 aminoacids, about 180 amino acids to about 220 amino acids, about 180 aminoacids to about 215 amino acids, about 180 amino acids to about 210 aminoacids, about 180 amino acids to about 205 amino acids, about 180 aminoacids to about 200 amino acids, about 180 amino acids to about 195 aminoacids, about 180 amino acids to about 190 amino acids, about 180 aminoacids to about 185 amino acids, about 185 amino acids to about 220 aminoacids, about 185 amino acids to about 215 amino acids, about 185 aminoacids to about 210 amino acids, about 185 amino acids to about 205 aminoacids, about 185 amino acids to about 200 amino acids, about 185 aminoacids to about 195 amino acids, about 185 amino acids to about 190 aminoacids, about 190 amino acids to about 220 amino acids, about 190 aminoacids to about 215 amino acids, about 190 amino acids to about 210 aminoacids, about 190 amino acids to about 205 amino acids, about 190 aminoacids to about 200 amino acids, about 190 amino acids to about 195 aminoacids, about 195 amino acids to about 220 amino acids, about 195 aminoacids to about 215 amino acids, about 195 amino acids to about 210 aminoacids, about 195 amino acids to about 205 amino acids, about 195 aminoacids to about 200 amino acids, about 200 amino acids to about 220 aminoacids, about 200 amino acids to about 215 amino acids, about 200 aminoacids to about 210 amino acids, about 200 amino acids to about 205 aminoacids, about 205 amino acids to about 220 amino acids, about 205 aminoacids to about 215 amino acids, about 205 amino acids to about 210 aminoacids, about 210 amino acids to about 220 amino acids, about 210 aminoacids to about 215 amino acids, or about 215 amino acids to about 220amino acids.

In some embodiments, the soluble tissue factor domain can comprise orconsist of a soluble wildtype human tissue factor (or any sequencetherefrom).

Linker Sequences

In some embodiments, the linker sequence can be a flexible linkersequence. Non-limiting examples of linker sequences that can be used aredescribed in Klein et al., Protein Engineering, Design & Selection Vol.27, No. 10, pp. 325-330, 2014; Priyanka et al., Protein Sci., 2013February; 22(2):153-167. In some examples, the linker sequence is asynthetic linker sequence.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein can include one, two, three, four, five, six, seven,eight, nine, or ten linker sequence(s) (e.g., the same or differentlinker sequences, e.g., any of the exemplary linker sequences describedherein or known in the art). In some embodiments of any of thesingle-chain chimeric polypeptides described herein can include one,two, three, four, five, six, seven, eight, nine, or ten linkersequence(s) (e.g., the same or different linker sequences, e.g., any ofthe exemplary linker sequences described herein or known in the art).

In some embodiments, a linker sequence can have a total length of 1amino acid to about 100 amino acids, 1 amino acid to about 90 aminoacids, 1 amino acid to about 80 amino acids, 1 amino acid to about 70amino acids, 1 amino acid to about 60 amino acids, 1 amino acid to about50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid toabout 40 amino acids, 1 amino acid to about 35 amino acids, 1 amino acidto about 30 amino acids, 1 amino acid to about 25 amino acids, 1 aminoacid to about 24 amino acids, 1 amino acid to about 22 amino acids, 1amino acid to about 20 amino acids, 1 amino acid to about 18 aminoacids, 1 amino acid to about 16 amino acids, 1 amino acid to about 14amino acids, 1 amino acid to about 12 amino acids, 1 amino acid to about10 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid toabout 6 amino acids, 1 amino acid to about 4 amino acids, about 2 aminoacids to about 100 amino acids, about 2 amino acids to about 90 aminoacids, about 2 amino acids to about 80 amino acids, about 2 amino acidsto about 70 amino acids, about 2 amino acids to about 60 amino acids,about 2 amino acids to about 50 amino acids, about 2 amino acids toabout 45 amino acids, about 2 amino acids to about 40 amino acids, about2 amino acids to about 35 amino acids, about 2 amino acids to about 30amino acids, about 2 amino acids to about 25 amino acids, about 2 aminoacids to about 24 amino acids, about 2 amino acids to about 22 aminoacids, about 2 amino acids to about 20 amino acids, about 2 amino acidsto about 18 amino acids, about 2 amino acids to about 16 amino acids,about 2 amino acids to about 14 amino acids, about 2 amino acids toabout 12 amino acids, about 2 amino acids to about 10 amino acids, about2 amino acids to about 8 amino acids, about 2 amino acids to about 6amino acids, about 2 amino acids to about 4 amino acids, about 4 aminoacids to about 100 amino acids, about 4 amino acids to about 90 aminoacids, about 4 amino acids to about 80 amino acids, about 4 amino acidsto about 70 amino acids, about 4 amino acids to about 60 amino acids,about 4 amino acids to about 50 amino acids, about 4 amino acids toabout 45 amino acids, about 4 amino acids to about 40 amino acids, about4 amino acids to about 35 amino acids, about 4 amino acids to about 30amino acids, about 4 amino acids to about 25 amino acids, about 4 aminoacids to about 24 amino acids, about 4 amino acids to about 22 aminoacids, about 4 amino acids to about 20 amino acids, about 4 amino acidsto about 18 amino acids, about 4 amino acids to about 16 amino acids,about 4 amino acids to about 14 amino acids, about 4 amino acids toabout 12 amino acids, about 4 amino acids to about 10 amino acids, about4 amino acids to about 8 amino acids, about 4 amino acids to about 6amino acids, about 6 amino acids to about 100 amino acids, about 6 aminoacids to about 90 amino acids, about 6 amino acids to about 80 aminoacids, about 6 amino acids to about 70 amino acids, about 6 amino acidsto about 60 amino acids, about 6 amino acids to about 50 amino acids,about 6 amino acids to about 45 amino acids, about 6 amino acids toabout 40 amino acids, about 6 amino acids to about 35 amino acids, about6 amino acids to about 30 amino acids, about 6 amino acids to about 25amino acids, about 6 amino acids to about 24 amino acids, about 6 aminoacids to about 22 amino acids, about 6 amino acids to about 20 aminoacids, about 6 amino acids to about 18 amino acids, about 6 amino acidsto about 16 amino acids, about 6 amino acids to about 14 amino acids,about 6 amino acids to about 12 amino acids, about 6 amino acids toabout 10 amino acids, about 6 amino acids to about 8 amino acids, about8 amino acids to about 100 amino acids, about 8 amino acids to about 90amino acids, about 8 amino acids to about 80 amino acids, about 8 aminoacids to about 70 amino acids, about 8 amino acids to about 60 aminoacids, about 8 amino acids to about 50 amino acids, about 8 amino acidsto about 45 amino acids, about 8 amino acids to about 40 amino acids,about 8 amino acids to about 35 amino acids, about 8 amino acids toabout 30 amino acids, about 8 amino acids to about 25 amino acids, about8 amino acids to about 24 amino acids, about 8 amino acids to about 22amino acids, about 8 amino acids to about 20 amino acids, about 8 aminoacids to about 18 amino acids, about 8 amino acids to about 16 aminoacids, about 8 amino acids to about 14 amino acids, about 8 amino acidsto about 12 amino acids, about 8 amino acids to about 10 amino acids,about 10 amino acids to about 100 amino acids, about 10 amino acids toabout 90 amino acids, about 10 amino acids to about 80 amino acids,about 10 amino acids to about 70 amino acids, about 10 amino acids toabout 60 amino acids, about 10 amino acids to about 50 amino acids,about 10 amino acids to about 45 amino acids, about 10 amino acids toabout 40 amino acids, about 10 amino acids to about 35 amino acids,about 10 amino acids to about 30 amino acids, about 10 amino acids toabout 25 amino acids, about 10 amino acids to about 24 amino acids,about 10 amino acids to about 22 amino acids, about 10 amino acids toabout 20 amino acids, about 10 amino acids to about 18 amino acids,about 10 amino acids to about 16 amino acids, about 10 amino acids toabout 14 amino acids, about 10 amino acids to about 12 amino acids,about 12 amino acids to about 100 amino acids, about 12 amino acids toabout 90 amino acids, about 12 amino acids to about 80 amino acids,about 12 amino acids to about 70 amino acids, about 12 amino acids toabout 60 amino acids, about 12 amino acids to about 50 amino acids,about 12 amino acids to about 45 amino acids, about 12 amino acids toabout 40 amino acids, about 12 amino acids to about 35 amino acids,about 12 amino acids to about 30 amino acids, about 12 amino acids toabout 25 amino acids, about 12 amino acids to about 24 amino acids,about 12 amino acids to about 22 amino acids, about 12 amino acids toabout 20 amino acids, about 12 amino acids to about 18 amino acids,about 12 amino acids to about 16 amino acids, about 12 amino acids toabout 14 amino acids, about 14 amino acids to about 100 amino acids,about 14 amino acids to about 90 amino acids, about 14 amino acids toabout 80 amino acids, about 14 amino acids to about 70 amino acids,about 14 amino acids to about 60 amino acids, about 14 amino acids toabout 50 amino acids, about 14 amino acids to about 45 amino acids,about 14 amino acids to about 40 amino acids, about 14 amino acids toabout 35 amino acids, about 14 amino acids to about 30 amino acids,about 14 amino acids to about 25 amino acids, about 14 amino acids toabout 24 amino acids, about 14 amino acids to about 22 amino acids,about 14 amino acids to about 20 amino acids, about 14 amino acids toabout 18 amino acids, about 14 amino acids to about 16 amino acids,about 16 amino acids to about 100 amino acids, about 16 amino acids toabout 90 amino acids, about 16 amino acids to about 80 amino acids,about 16 amino acids to about 70 amino acids, about 16 amino acids toabout 60 amino acids, about 16 amino acids to about 50 amino acids,about 16 amino acids to about 45 amino acids, about 16 amino acids toabout 40 amino acids, about 16 amino acids to about 35 amino acids,about 16 amino acids to about 30 amino acids, about 16 amino acids toabout 25 amino acids, about 16 amino acids to about 24 amino acids,about 16 amino acids to about 22 amino acids, about 16 amino acids toabout 20 amino acids, about 16 amino acids to about 18 amino acids,about 18 amino acids to about 100 amino acids, about 18 amino acids toabout 90 amino acids, about 18 amino acids to about 80 amino acids,about 18 amino acids to about 70 amino acids, about 18 amino acids toabout 60 amino acids, about 18 amino acids to about 50 amino acids,about 18 amino acids to about 45 amino acids, about 18 amino acids toabout 40 amino acids, about 18 amino acids to about 35 amino acids,about 18 amino acids to about 30 amino acids, about 18 amino acids toabout 25 amino acids, about 18 amino acids to about 24 amino acids,about 18 amino acids to about 22 amino acids, about 18 amino acids toabout 20 amino acids, about 20 amino acids to about 100 amino acids,about 20 amino acids to about 90 amino acids, about 20 amino acids toabout 80 amino acids, about 20 amino acids to about 70 amino acids,about 20 amino acids to about 60 amino acids, about 20 amino acids toabout 50 amino acids, about 20 amino acids to about 45 amino acids,about 20 amino acids to about 40 amino acids, about 20 amino acids toabout 35 amino acids, about 20 amino acids to about 30 amino acids,about 20 amino acids to about 25 amino acids, about 20 amino acids toabout 24 amino acids, about 20 amino acids to about 22 amino acids,about 22 amino acids to about 100 amino acids, about 22 amino acids toabout 90 amino acids, about 22 amino acids to about 80 amino acids,about 22 amino acids to about 70 amino acids, about 22 amino acids toabout 60 amino acids, about 22 amino acids to about 50 amino acids,about 22 amino acids to about 45 amino acids, about 22 amino acids toabout 40 amino acids, about 22 amino acids to about 35 amino acids,about 22 amino acids to about 30 amino acids, about 22 amino acids toabout 25 amino acids, about 22 amino acids to about 24 amino acids,about 25 amino acids to about 100 amino acids, about 25 amino acids toabout 90 amino acids, about 25 amino acids to about 80 amino acids,about 25 amino acids to about 70 amino acids, about 25 amino acids toabout 60 amino acids, about 25 amino acids to about 50 amino acids,about 25 amino acids to about 45 amino acids, about 25 amino acids toabout 40 amino acids, about 25 amino acids to about 35 amino acids,about 25 amino acids to about 30 amino acids, about 30 amino acids toabout 100 amino acids, about 30 amino acids to about 90 amino acids,about 30 amino acids to about 80 amino acids, about 30 amino acids toabout 70 amino acids, about 30 amino acids to about 60 amino acids,about 30 amino acids to about 50 amino acids, about 30 amino acids toabout 45 amino acids, about 30 amino acids to about 40 amino acids,about 30 amino acids to about 35 amino acids, about 35 amino acids toabout 100 amino acids, about 35 amino acids to about 90 amino acids,about 35 amino acids to about 80 amino acids, about 35 amino acids toabout 70 amino acids, about 35 amino acids to about 60 amino acids,about 35 amino acids to about 50 amino acids, about 35 amino acids toabout 45 amino acids, about 35 amino acids to about 40 amino acids,about 40 amino acids to about 100 amino acids, about 40 amino acids toabout 90 amino acids, about 40 amino acids to about 80 amino acids,about 40 amino acids to about 70 amino acids, about 40 amino acids toabout 60 amino acids, about 40 amino acids to about 50 amino acids,about 40 amino acids to about 45 amino acids, about 45 amino acids toabout 100 amino acids, about 45 amino acids to about 90 amino acids,about 45 amino acids to about 80 amino acids, about 45 amino acids toabout 70 amino acids, about 45 amino acids to about 60 amino acids,about 45 amino acids to about 50 amino acids, about 50 amino acids toabout 100 amino acids, about 50 amino acids to about 90 amino acids,about 50 amino acids to about 80 amino acids, about 50 amino acids toabout 70 amino acids, about 50 amino acids to about 60 amino acids,about 60 amino acids to about 100 amino acids, about 60 amino acids toabout 90 amino acids, about 60 amino acids to about 80 amino acids,about 60 amino acids to about 70 amino acids, about 70 amino acids toabout 100 amino acids, about 70 amino acids to about 90 amino acids,about 70 amino acids to about 80 amino acids, about 80 amino acids toabout 100 amino acids, about 80 amino acids to about 90 amino acids, orabout 90 amino acids to about 100 amino acids.

In some embodiments, the linker is rich in glycine (Gly or G) residues.In some embodiments, the linker is rich in serine (Ser or S) residues.In some embodiments, the linker is rich in glycine and serine residues.In some embodiments, the linker has one or more glycine-serine residuepairs (GS), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. Insome embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS)sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGSsequences. In some embodiments, the linker has one or moreGly-Gly-Gly-Gly-Ser (GGGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 or more GGGGS sequences. In some embodiments, the linker has oneor more Gly-Gly-Ser-Gly (GGSG) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more GGSG sequences.

In some embodiments, the linker sequence can comprise or consist ofGGGGSGGGGSGGGGS (SEQ ID NO: 13). In some embodiments, the linkersequence can be encoded by a nucleic acid comprising or consisting of:GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 14). In someembodiments, the linker sequence can comprise or consist of: GGGSGGGS(SEQ ID NO: 15).

Target-Binding Domains

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain, the secondtarget-binding domain, and/or the additional one or more target-bindingdomains can be an antigen-binding domain (e.g., any of the exemplaryantigen-binding domains described herein or known in the art), a solubleinterleukin or cytokine protein (e.g., any of the exemplary solubleinterleukin proteins or soluble cytokine proteins described herein), anda soluble interleukin or cytokine receptor (e.g., any of the exemplarysoluble interleukin receptors or soluble cytokine receptors describedherein).

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, one or more of the first target-binding domain (e.g.,any of the exemplary first target binding domains described herein orknown in the art), the second target-binding domain (e.g., any of theexemplary second target binding domains described herein or known in theart), and the one or more additional target binding domains can each,independently, bind specifically to a target selected from the group of:bind specifically to a target selected from the group consisting of:CD16a, CD28, CD3 (e.g., one or more of CD3α, CD3β, CD3δ, CD3ε, andCD3γ), CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4,IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα,CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET,EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,CEACAM5, a UL16-binding protein (e.g., ULBP1, ULBP2, ULBP3, ULBP4,ULBP5, and ULBP6), HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD,a ligand of TGF-β receptor II (TGF-βRII), a ligand of TGF-βRIII, aligand of DNAM1, a ligand of NKp46, a ligand of NKp44, a ligand ofNKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII,a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, areceptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptorfor IL-10, a receptor for IL-12, a receptor for IL-15, a receptor forIL-17, a receptor for IL-18, a receptor for IL-21, a receptor forPDGF-DD, a receptor for stem cell factor (SCF), a receptor for stemcell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, areceptor for MICB, a receptor for a ULP16-binding protein, a receptorfor CD155, a receptor for CD122, and a receptor for CD28.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain, the secondtarget-binding domain, and/or the one or more additional target-bindingdomains can each independent have a total number of amino acids of about5 amino acids to about 1000 amino acids, about 5 amino acids to about950 amino acids, about 5 amino acids to about 900 amino acids, about 5amino acids to about 850 amino acids, about 5 amino acids to about 800amino acids, about 5 amino acids to about 750 amino acids, about 5 aminoacids to about 700 amino acids, about 5 amino acids to about 650 aminoacids, about 5 amino acids to about 600 amino acids, about 5 amino acidsto about 550 amino acids, about 5 amino acids to about 500 amino acids,about 5 amino acids to about 450 amino acids, about 5 amino acids toabout 400 amino acids, about 5 amino acids to about 350 amino acids,about 5 amino acids to about 300 amino acids, about 5 amino acids toabout 280 amino acids, about 5 amino acids to about 260 amino acids,about 5 amino acids to about 240 amino acids, about 5 amino acids toabout 220 amino acids, about 5 amino acids to about 200 amino acids,about 5 amino acids to about 195 amino acids, about 5 amino acids toabout 190 amino acids, about 5 amino acids to about 185 amino acids,about 5 amino acids to about 180 amino acids, about 5 amino acids toabout 175 amino acids, about 5 amino acids to about 170 amino acids,about 5 amino acids to about 165 amino acids, about 5 amino acids toabout 160 amino acids, about 5 amino acids to about 155 amino acids,about 5 amino acids to about 150 amino acids, about 5 amino acids toabout 145 amino acids, about 5 amino acids to about 140 amino acids,about 5 amino acids to about 135 amino acids, about 5 amino acids toabout 130 amino acids, about 5 amino acids to about 125 amino acids,about 5 amino acids to about 120 amino acids, about 5 amino acids toabout 115 amino acids, about 5 amino acids to about 110 amino acids,about 5 amino acids to about 105 amino acids, about 5 amino acids toabout 100 amino acids, about 5 amino acids to about 95 amino acids,about 5 amino acids to about 90 amino acids, about 5 amino acids toabout 85 amino acids, about 5 amino acids to about 80 amino acids, about5 amino acids to about 75 amino acids, about 5 amino acids to about 70amino acids, about 5 amino acids to about 65 amino acids, about 5 aminoacids to about 60 amino acids, about 5 amino acids to about 55 aminoacids, about 5 amino acids to about 50 amino acids, about 5 amino acidsto about 45 amino acids, about 5 amino acids to about 40 amino acids,about 5 amino acids to about 35 amino acids, about 5 amino acids toabout 30 amino acids, about 5 amino acids to about 25 amino acids, about5 amino acids to about 20 amino acids, about 5 amino acids to about 15amino acids, about 5 amino acids to about 10 amino acids, about 10 aminoacids to about 1000 amino acids, about 10 amino acids to about 950 aminoacids, about 10 amino acids to about 900 amino acids, about 10 aminoacids to about 850 amino acids, about 10 amino acids to about 800 aminoacids, about 10 amino acids to about 750 amino acids, about 10 aminoacids to about 700 amino acids, about 10 amino acids to about 650 aminoacids, about 10 amino acids to about 600 amino acids, about 10 aminoacids to about 550 amino acids, about 10 amino acids to about 500 aminoacids, about 10 amino acids to about 450 amino acids, about 10 aminoacids to about 400 amino acids, about 10 amino acids to about 350 aminoacids, about 10 amino acids to about 300 amino acids, about 10 aminoacids to about 280 amino acids, about 10 amino acids to about 260 aminoacids, about 10 amino acids to about 240 amino acids, about 10 aminoacids to about 220 amino acids, about 10 amino acids to about 200 aminoacids, about 10 amino acids to about 195 amino acids, about 10 aminoacids to about 190 amino acids, about 10 amino acids to about 185 aminoacids, about 10 amino acids to about 180 amino acids, about 10 aminoacids to about 175 amino acids, about 10 amino acids to about 170 aminoacids, about 10 amino acids to about 165 amino acids, about 10 aminoacids to about 160 amino acids, about 10 amino acids to about 155 aminoacids, about 10 amino acids to about 150 amino acids, about 10 aminoacids to about 145 amino acids, about 10 amino acids to about 140 aminoacids, about 10 amino acids to about 135 amino acids, about 10 aminoacids to about 130 amino acids, about 10 amino acids to about 125 aminoacids, about 10 amino acids to about 120 amino acids, about 10 aminoacids to about 115 amino acids, about 10 amino acids to about 110 aminoacids, about 10 amino acids to about 105 amino acids, about 10 aminoacids to about 100 amino acids, about 10 amino acids to about 95 aminoacids, about 10 amino acids to about 90 amino acids, about 10 aminoacids to about 85 amino acids, about 10 amino acids to about 80 aminoacids, about 10 amino acids to about 75 amino acids, about 10 aminoacids to about 70 amino acids, about 10 amino acids to about 65 aminoacids, about 10 amino acids to about 60 amino acids, about 10 aminoacids to about 55 amino acids, about 10 amino acids to about 50 aminoacids, about 10 amino acids to about 45 amino acids, about 10 aminoacids to about 40 amino acids, about 10 amino acids to about 35 aminoacids, about 10 amino acids to about 30 amino acids, about 10 aminoacids to about 25 amino acids, about 10 amino acids to about 20 aminoacids, about 10 amino acids to about 15 amino acids, about 15 aminoacids to about 1000 amino acids, about 15 amino acids to about 950 aminoacids, about 15 amino acids to about 900 amino acids, about 15 aminoacids to about 850 amino acids, about 15 amino acids to about 800 aminoacids, about 15 amino acids to about 750 amino acids, about 15 aminoacids to about 700 amino acids, about 15 amino acids to about 650 aminoacids, about 15 amino acids to about 600 amino acids, about 15 aminoacids to about 550 amino acids, about 15 amino acids to about 500 aminoacids, about 15 amino acids to about 450 amino acids, about 15 aminoacids to about 400 amino acids, about 15 amino acids to about 350 aminoacids, about 15 amino acids to about 300 amino acids, about 15 aminoacids to about 280 amino acids, about 15 amino acids to about 260 aminoacids, about 15 amino acids to about 240 amino acids, about 15 aminoacids to about 220 amino acids, about 15 amino acids to about 200 aminoacids, about 15 amino acids to about 195 amino acids, about 15 aminoacids to about 190 amino acids, about 15 amino acids to about 185 aminoacids, about 15 amino acids to about 180 amino acids, about 15 aminoacids to about 175 amino acids, about 15 amino acids to about 170 aminoacids, about 15 amino acids to about 165 amino acids, about 15 aminoacids to about 160 amino acids, about 15 amino acids to about 155 aminoacids, about 15 amino acids to about 150 amino acids, about 15 aminoacids to about 145 amino acids, about 15 amino acids to about 140 aminoacids, about 15 amino acids to about 135 amino acids, about 15 aminoacids to about 130 amino acids, about 15 amino acids to about 125 aminoacids, about 15 amino acids to about 120 amino acids, about 15 aminoacids to about 115 amino acids, about 15 amino acids to about 110 aminoacids, about 15 amino acids to about 105 amino acids, about 15 aminoacids to about 100 amino acids, about 15 amino acids to about 95 aminoacids, about 15 amino acids to about 90 amino acids, about 15 aminoacids to about 85 amino acids, about 15 amino acids to about 80 aminoacids, about 15 amino acids to about 75 amino acids, about 15 aminoacids to about 70 amino acids, about 15 amino acids to about 65 aminoacids, about 15 amino acids to about 60 amino acids, about 15 aminoacids to about 55 amino acids, about 15 amino acids to about 50 aminoacids, about 15 amino acids to about 45 amino acids, about 15 aminoacids to about 40 amino acids, about 15 amino acids to about 35 aminoacids, about 15 amino acids to about 30 amino acids, about 15 aminoacids to about 25 amino acids, about 15 amino acids to about 20 aminoacids, about 20 amino acids to about 1000 amino acids, about 20 aminoacids to about 950 amino acids, about 20 amino acids to about 900 aminoacids, about 20 amino acids to about 850 amino acids, about 20 aminoacids to about 800 amino acids, about 20 amino acids to about 750 aminoacids, about 20 amino acids to about 700 amino acids, about 20 aminoacids to about 650 amino acids, about 20 amino acids to about 600 aminoacids, about 20 amino acids to about 550 amino acids, about 20 aminoacids to about 500 amino acids, about 20 amino acids to about 450 aminoacids, about 20 amino acids to about 400 amino acids, about 20 aminoacids to about 350 amino acids, about 20 amino acids to about 300 aminoacids, about 20 amino acids to about 280 amino acids, about 20 aminoacids to about 260 amino acids, about 20 amino acids to about 240 aminoacids, about 20 amino acids to about 220 amino acids, about 20 aminoacids to about 200 amino acids, about 20 amino acids to about 195 aminoacids, about 20 amino acids to about 190 amino acids, about 20 aminoacids to about 185 amino acids, about 20 amino acids to about 180 aminoacids, about 20 amino acids to about 175 amino acids, about 20 aminoacids to about 170 amino acids, about 20 amino acids to about 165 aminoacids, about 20 amino acids to about 160 amino acids, about 20 aminoacids to about 155 amino acids, about 20 amino acids to about 150 aminoacids, about 20 amino acids to about 145 amino acids, about 20 aminoacids to about 140 amino acids, about 20 amino acids to about 135 aminoacids, about 20 amino acids to about 130 amino acids, about 20 aminoacids to about 125 amino acids, about 20 amino acids to about 120 aminoacids, about 20 amino acids to about 115 amino acids, about 20 aminoacids to about 110 amino acids, about 20 amino acids to about 105 aminoacids, about 20 amino acids to about 100 amino acids, about 20 aminoacids to about 95 amino acids, about 20 amino acids to about 90 aminoacids, about 20 amino acids to about 85 amino acids, about 20 aminoacids to about 80 amino acids, about 20 amino acids to about 75 aminoacids, about 20 amino acids to about 70 amino acids, about 20 aminoacids to about 65 amino acids, about 20 amino acids to about 60 aminoacids, about 20 amino acids to about 55 amino acids, about 20 aminoacids to about 50 amino acids, about 20 amino acids to about 45 aminoacids, about 20 amino acids to about 40 amino acids, about 20 aminoacids to about 35 amino acids, about 20 amino acids to about 30 aminoacids, about 20 amino acids to about 25 amino acids, about 25 aminoacids to about 1000 amino acids, about 25 amino acids to about 950 aminoacids, about 25 amino acids to about 900 amino acids, about 25 aminoacids to about 850 amino acids, about 25 amino acids to about 800 aminoacids, about 25 amino acids to about 750 amino acids, about 25 aminoacids to about 700 amino acids, about 25 amino acids to about 650 aminoacids, about 25 amino acids to about 600 amino acids, about 25 aminoacids to about 550 amino acids, about 25 amino acids to about 500 aminoacids, about 25 amino acids to about 450 amino acids, about 25 aminoacids to about 400 amino acids, about 25 amino acids to about 350 aminoacids, about 25 amino acids to about 300 amino acids, about 25 aminoacids to about 280 amino acids, about 25 amino acids to about 260 aminoacids, about 25 amino acids to about 240 amino acids, about 25 aminoacids to about 220 amino acids, about 25 amino acids to about 200 aminoacids, about 25 amino acids to about 195 amino acids, about 25 aminoacids to about 190 amino acids, about 25 amino acids to about 185 aminoacids, about 25 amino acids to about 180 amino acids, about 25 aminoacids to about 175 amino acids, about 25 amino acids to about 170 aminoacids, about 25 amino acids to about 165 amino acids, about 25 aminoacids to about 160 amino acids, about 25 amino acids to about 155 aminoacids, about 25 amino acids to about 150 amino acids, about 25 aminoacids to about 145 amino acids, about 25 amino acids to about 140 aminoacids, about 25 amino acids to about 135 amino acids, about 25 aminoacids to about 130 amino acids, about 25 amino acids to about 125 aminoacids, about 25 amino acids to about 120 amino acids, about 25 aminoacids to about 115 amino acids, about 25 amino acids to about 110 aminoacids, about 25 amino acids to about 105 amino acids, about 25 aminoacids to about 100 amino acids, about 25 amino acids to about 95 aminoacids, about 25 amino acids to about 90 amino acids, about 25 aminoacids to about 85 amino acids, about 25 amino acids to about 80 aminoacids, about 25 amino acids to about 75 amino acids, about 25 aminoacids to about 70 amino acids, about 25 amino acids to about 65 aminoacids, about 25 amino acids to about 60 amino acids, about 25 aminoacids to about 55 amino acids, about 25 amino acids to about 50 aminoacids, about 25 amino acids to about 45 amino acids, about 25 aminoacids to about 40 amino acids, about 25 amino acids to about 35 aminoacids, about 25 amino acids to about 30 amino acids, about 30 aminoacids to about 1000 amino acids, about 30 amino acids to about 950 aminoacids, about 30 amino acids to about 900 amino acids, about 30 aminoacids to about 850 amino acids, about 30 amino acids to about 800 aminoacids, about 30 amino acids to about 750 amino acids, about 30 aminoacids to about 700 amino acids, about 30 amino acids to about 650 aminoacids, about 30 amino acids to about 600 amino acids, about 30 aminoacids to about 550 amino acids, about 30 amino acids to about 500 aminoacids, about 30 amino acids to about 450 amino acids, about 30 aminoacids to about 400 amino acids, about 30 amino acids to about 350 aminoacids, about 30 amino acids to about 300 amino acids, about 30 aminoacids to about 280 amino acids, about 30 amino acids to about 260 aminoacids, about 30 amino acids to about 240 amino acids, about 30 aminoacids to about 220 amino acids, about 30 amino acids to about 200 aminoacids, about 30 amino acids to about 195 amino acids, about 30 aminoacids to about 190 amino acids, about 30 amino acids to about 185 aminoacids, about 30 amino acids to about 180 amino acids, about 30 aminoacids to about 175 amino acids, about 30 amino acids to about 170 aminoacids, about 30 amino acids to about 165 amino acids, about 30 aminoacids to about 160 amino acids, about 30 amino acids to about 155 aminoacids, about 30 amino acids to about 150 amino acids, about 30 aminoacids to about 145 amino acids, about 30 amino acids to about 140 aminoacids, about 30 amino acids to about 135 amino acids, about 30 aminoacids to about 130 amino acids, about 30 amino acids to about 125 aminoacids, about 30 amino acids to about 120 amino acids, about 30 aminoacids to about 115 amino acids, about 30 amino acids to about 110 aminoacids, about 30 amino acids to about 105 amino acids, about 30 aminoacids to about 100 amino acids, about 30 amino acids to about 95 aminoacids, about 30 amino acids to about 90 amino acids, about 30 aminoacids to about 85 amino acids, about 30 amino acids to about 80 aminoacids, about 30 amino acids to about 75 amino acids, about 30 aminoacids to about 70 amino acids, about 30 amino acids to about 65 aminoacids, about 30 amino acids to about 60 amino acids, about 30 aminoacids to about 55 amino acids, about 30 amino acids to about 50 aminoacids, about 30 amino acids to about 45 amino acids, about 30 aminoacids to about 40 amino acids, about 30 amino acids to about 35 aminoacids, about 35 amino acids to about 1000 amino acids, about 35 aminoacids to about 950 amino acids, about 35 amino acids to about 900 aminoacids, about 35 amino acids to about 850 amino acids, about 35 aminoacids to about 800 amino acids, about 35 amino acids to about 750 aminoacids, about 35 amino acids to about 700 amino acids, about 35 aminoacids to about 650 amino acids, about 35 amino acids to about 600 aminoacids, about 35 amino acids to about 550 amino acids, about 35 aminoacids to about 500 amino acids, about 35 amino acids to about 450 aminoacids, about 35 amino acids to about 400 amino acids, about 35 aminoacids to about 350 amino acids, about 35 amino acids to about 300 aminoacids, about 35 amino acids to about 280 amino acids, about 35 aminoacids to about 260 amino acids, about 35 amino acids to about 240 aminoacids, about 35 amino acids to about 220 amino acids, about 35 aminoacids to about 200 amino acids, about 35 amino acids to about 195 aminoacids, about 35 amino acids to about 190 amino acids, about 35 aminoacids to about 185 amino acids, about 35 amino acids to about 180 aminoacids, about 35 amino acids to about 175 amino acids, about 35 aminoacids to about 170 amino acids, about 35 amino acids to about 165 aminoacids, about 35 amino acids to about 160 amino acids, about 35 aminoacids to about 155 amino acids, about 35 amino acids to about 150 aminoacids, about 35 amino acids to about 145 amino acids, about 35 aminoacids to about 140 amino acids, about 35 amino acids to about 135 aminoacids, about 35 amino acids to about 130 amino acids, about 35 aminoacids to about 125 amino acids, about 35 amino acids to about 120 aminoacids, about 35 amino acids to about 115 amino acids, about 35 aminoacids to about 110 amino acids, about 35 amino acids to about 105 aminoacids, about 35 amino acids to about 100 amino acids, about 35 aminoacids to about 95 amino acids, about 35 amino acids to about 90 aminoacids, about 35 amino acids to about 85 amino acids, about 35 aminoacids to about 80 amino acids, about 35 amino acids to about 75 aminoacids, about 35 amino acids to about 70 amino acids, about 35 aminoacids to about 65 amino acids, about 35 amino acids to about 60 aminoacids, about 35 amino acids to about 55 amino acids, about 35 aminoacids to about 50 amino acids, about 35 amino acids to about 45 aminoacids, about 35 amino acids to about 40 amino acids, about 40 aminoacids to about 1000 amino acids, about 40 amino acids to about 950 aminoacids, about 40 amino acids to about 900 amino acids, about 40 aminoacids to about 850 amino acids, about 40 amino acids to about 800 aminoacids, about 40 amino acids to about 750 amino acids, about 40 aminoacids to about 700 amino acids, about 40 amino acids to about 650 aminoacids, about 40 amino acids to about 600 amino acids, about 40 aminoacids to about 550 amino acids, about 40 amino acids to about 500 aminoacids, about 40 amino acids to about 450 amino acids, about 40 aminoacids to about 400 amino acids, about 40 amino acids to about 350 aminoacids, about 40 amino acids to about 300 amino acids, about 40 aminoacids to about 280 amino acids, about 40 amino acids to about 260 aminoacids, about 40 amino acids to about 240 amino acids, about 40 aminoacids to about 220 amino acids, about 40 amino acids to about 200 aminoacids, about 40 amino acids to about 195 amino acids, about 40 aminoacids to about 190 amino acids, about 40 amino acids to about 185 aminoacids, about 40 amino acids to about 180 amino acids, about 40 aminoacids to about 175 amino acids, about 40 amino acids to about 170 aminoacids, about 40 amino acids to about 165 amino acids, about 40 aminoacids to about 160 amino acids, about 40 amino acids to about 155 aminoacids, about 40 amino acids to about 150 amino acids, about 40 aminoacids to about 145 amino acids, about 40 amino acids to about 140 aminoacids, about 40 amino acids to about 135 amino acids, about 40 aminoacids to about 130 amino acids, about 40 amino acids to about 125 aminoacids, about 40 amino acids to about 120 amino acids, about 40 aminoacids to about 115 amino acids, about 40 amino acids to about 110 aminoacids, about 40 amino acids to about 105 amino acids, about 40 aminoacids to about 100 amino acids, about 40 amino acids to about 95 aminoacids, about 40 amino acids to about 90 amino acids, about 40 aminoacids to about 85 amino acids, about 40 amino acids to about 80 aminoacids, about 40 amino acids to about 75 amino acids, about 40 aminoacids to about 70 amino acids, about 40 amino acids to about 65 aminoacids, about 40 amino acids to about 60 amino acids, about 40 aminoacids to about 55 amino acids, about 40 amino acids to about 50 aminoacids, about 40 amino acids to about 45 amino acids, about 45 aminoacids to about 1000 amino acids, about 45 amino acids to about 950 aminoacids, about 45 amino acids to about 900 amino acids, about 45 aminoacids to about 850 amino acids, about 45 amino acids to about 800 aminoacids, about 45 amino acids to about 750 amino acids, about 45 aminoacids to about 700 amino acids, about 45 amino acids to about 650 aminoacids, about 45 amino acids to about 600 amino acids, about 45 aminoacids to about 550 amino acids, about 45 amino acids to about 500 aminoacids, about 45 amino acids to about 450 amino acids, about 45 aminoacids to about 400 amino acids, about 45 amino acids to about 350 aminoacids, about 45 amino acids to about 300 amino acids, about 45 aminoacids to about 280 amino acids, about 45 amino acids to about 260 aminoacids, about 45 amino acids to about 240 amino acids, about 45 aminoacids to about 220 amino acids, about 45 amino acids to about 200 aminoacids, about 45 amino acids to about 195 amino acids, about 45 aminoacids to about 190 amino acids, about 45 amino acids to about 185 aminoacids, about 45 amino acids to about 180 amino acids, about 45 aminoacids to about 175 amino acids, about 45 amino acids to about 170 aminoacids, about 45 amino acids to about 165 amino acids, about 45 aminoacids to about 160 amino acids, about 45 amino acids to about 155 aminoacids, about 45 amino acids to about 150 amino acids, about 45 aminoacids to about 145 amino acids, about 45 amino acids to about 140 aminoacids, about 45 amino acids to about 135 amino acids, about 45 aminoacids to about 130 amino acids, about 45 amino acids to about 125 aminoacids, about 45 amino acids to about 120 amino acids, about 45 aminoacids to about 115 amino acids, about 45 amino acids to about 110 aminoacids, about 45 amino acids to about 105 amino acids, about 45 aminoacids to about 100 amino acids, about 45 amino acids to about 95 aminoacids, about 45 amino acids to about 90 amino acids, about 45 aminoacids to about 85 amino acids, about 45 amino acids to about 80 aminoacids, about 45 amino acids to about 75 amino acids, about 45 aminoacids to about 70 amino acids, about 45 amino acids to about 65 aminoacids, about 45 amino acids to about 60 amino acids, about 45 aminoacids to about 55 amino acids, about 45 amino acids to about 50 aminoacids, about 50 amino acids to about 1000 amino acids, about 50 aminoacids to about 950 amino acids, about 50 amino acids to about 900 aminoacids, about 50 amino acids to about 850 amino acids, about 50 aminoacids to about 800 amino acids, about 50 amino acids to about 750 aminoacids, about 50 amino acids to about 700 amino acids, about 50 aminoacids to about 650 amino acids, about 50 amino acids to about 600 aminoacids, about 50 amino acids to about 550 amino acids, about 50 aminoacids to about 500 amino acids, about 50 amino acids to about 450 aminoacids, about 50 amino acids to about 400 amino acids, about 50 aminoacids to about 350 amino acids, about 50 amino acids to about 300 aminoacids, about 50 amino acids to about 280 amino acids, about 50 aminoacids to about 260 amino acids, about 50 amino acids to about 240 aminoacids, about 50 amino acids to about 220 amino acids, about 50 aminoacids to about 200 amino acids, about 50 amino acids to about 195 aminoacids, about 50 amino acids to about 190 amino acids, about 50 aminoacids to about 185 amino acids, about 50 amino acids to about 180 aminoacids, about 50 amino acids to about 175 amino acids, about 50 aminoacids to about 170 amino acids, about 50 amino acids to about 165 aminoacids, about 50 amino acids to about 160 amino acids, about 50 aminoacids to about 155 amino acids, about 50 amino acids to about 150 aminoacids, about 50 amino acids to about 145 amino acids, about 50 aminoacids to about 140 amino acids, about 50 amino acids to about 135 aminoacids, about 50 amino acids to about 130 amino acids, about 50 aminoacids to about 125 amino acids, about 50 amino acids to about 120 aminoacids, about 50 amino acids to about 115 amino acids, about 50 aminoacids to about 110 amino acids, about 50 amino acids to about 105 aminoacids, about 50 amino acids to about 100 amino acids, about 50 aminoacids to about 95 amino acids, about 50 amino acids to about 90 aminoacids, about 50 amino acids to about 85 amino acids, about 50 aminoacids to about 80 amino acids, about 50 amino acids to about 75 aminoacids, about 50 amino acids to about 70 amino acids, about 50 aminoacids to about 65 amino acids, about 50 amino acids to about 60 aminoacids, about 50 amino acids to about 55 amino acids, about 55 aminoacids to about 1000 amino acids, about 55 amino acids to about 950 aminoacids, about 55 amino acids to about 900 amino acids, about 55 aminoacids to about 850 amino acids, about 55 amino acids to about 800 aminoacids, about 55 amino acids to about 750 amino acids, about 55 aminoacids to about 700 amino acids, about 55 amino acids to about 650 aminoacids, about 55 amino acids to about 600 amino acids, about 55 aminoacids to about 550 amino acids, about 55 amino acids to about 500 aminoacids, about 55 amino acids to about 450 amino acids, about 55 aminoacids to about 400 amino acids, about 55 amino acids to about 350 aminoacids, about 55 amino acids to about 300 amino acids, about 55 aminoacids to about 280 amino acids, about 55 amino acids to about 260 aminoacids, about 55 amino acids to about 240 amino acids, about 55 aminoacids to about 220 amino acids, about 55 amino acids to about 200 aminoacids, about 55 amino acids to about 195 amino acids, about 55 aminoacids to about 190 amino acids, about 55 amino acids to about 185 aminoacids, about 55 amino acids to about 180 amino acids, about 55 aminoacids to about 175 amino acids, about 55 amino acids to about 170 aminoacids, about 55 amino acids to about 165 amino acids, about 55 aminoacids to about 160 amino acids, about 55 amino acids to about 155 aminoacids, about 55 amino acids to about 150 amino acids, about 55 aminoacids to about 145 amino acids, about 55 amino acids to about 140 aminoacids, about 55 amino acids to about 135 amino acids, about 55 aminoacids to about 130 amino acids, about 55 amino acids to about 125 aminoacids, about 55 amino acids to about 120 amino acids, about 55 aminoacids to about 115 amino acids, about 55 amino acids to about 110 aminoacids, about 55 amino acids to about 105 amino acids, about 55 aminoacids to about 100 amino acids, about 55 amino acids to about 95 aminoacids, about 55 amino acids to about 90 amino acids, about 55 aminoacids to about 85 amino acids, about 55 amino acids to about 80 aminoacids, about 55 amino acids to about 75 amino acids, about 55 aminoacids to about 70 amino acids, about 55 amino acids to about 65 aminoacids, about 55 amino acids to about 60 amino acids, about 60 aminoacids to about 1000 amino acids, about 60 amino acids to about 950 aminoacids, about 60 amino acids to about 900 amino acids, about 60 aminoacids to about 850 amino acids, about 60 amino acids to about 800 aminoacids, about 60 amino acids to about 750 amino acids, about 60 aminoacids to about 700 amino acids, about 60 amino acids to about 650 aminoacids, about 60 amino acids to about 600 amino acids, about 60 aminoacids to about 550 amino acids, about 60 amino acids to about 500 aminoacids, about 60 amino acids to about 450 amino acids, about 60 aminoacids to about 400 amino acids, about 60 amino acids to about 350 aminoacids, about 60 amino acids to about 300 amino acids, about 60 aminoacids to about 280 amino acids, about 60 amino acids to about 260 aminoacids, about 60 amino acids to about 240 amino acids, about 60 aminoacids to about 220 amino acids, about 60 amino acids to about 200 aminoacids, about 60 amino acids to about 195 amino acids, about 60 aminoacids to about 190 amino acids, about 60 amino acids to about 185 aminoacids, about 60 amino acids to about 180 amino acids, about 60 aminoacids to about 175 amino acids, about 60 amino acids to about 170 aminoacids, about 60 amino acids to about 165 amino acids, about 60 aminoacids to about 160 amino acids, about 60 amino acids to about 155 aminoacids, about 60 amino acids to about 150 amino acids, about 60 aminoacids to about 145 amino acids, about 60 amino acids to about 140 aminoacids, about 60 amino acids to about 135 amino acids, about 60 aminoacids to about 130 amino acids, about 60 amino acids to about 125 aminoacids, about 60 amino acids to about 120 amino acids, about 60 aminoacids to about 115 amino acids, about 60 amino acids to about 110 aminoacids, about 60 amino acids to about 105 amino acids, about 60 aminoacids to about 100 amino acids, about 60 amino acids to about 95 aminoacids, about 60 amino acids to about 90 amino acids, about 60 aminoacids to about 85 amino acids, about 60 amino acids to about 80 aminoacids, about 60 amino acids to about 75 amino acids, about 60 aminoacids to about 70 amino acids, about 60 amino acids to about 65 aminoacids, about 65 amino acids to about 1000 amino acids, about 65 aminoacids to about 950 amino acids, about 65 amino acids to about 900 aminoacids, about 65 amino acids to about 850 amino acids, about 65 aminoacids to about 800 amino acids, about 65 amino acids to about 750 aminoacids, about 65 amino acids to about 700 amino acids, about 65 aminoacids to about 650 amino acids, about 65 amino acids to about 600 aminoacids, about 65 amino acids to about 550 amino acids, about 65 aminoacids to about 500 amino acids, about 65 amino acids to about 450 aminoacids, about 65 amino acids to about 400 amino acids, about 65 aminoacids to about 350 amino acids, about 65 amino acids to about 300 aminoacids, about 65 amino acids to about 280 amino acids, about 65 aminoacids to about 260 amino acids, about 65 amino acids to about 240 aminoacids, about 65 amino acids to about 220 amino acids, about 65 aminoacids to about 200 amino acids, about 65 amino acids to about 195 aminoacids, about 65 amino acids to about 190 amino acids, about 65 aminoacids to about 185 amino acids, about 65 amino acids to about 180 aminoacids, about 65 amino acids to about 175 amino acids, about 65 aminoacids to about 170 amino acids, about 65 amino acids to about 165 aminoacids, about 65 amino acids to about 160 amino acids, about 65 aminoacids to about 155 amino acids, about 65 amino acids to about 150 aminoacids, about 65 amino acids to about 145 amino acids, about 65 aminoacids to about 140 amino acids, about 65 amino acids to about 135 aminoacids, about 65 amino acids to about 130 amino acids, about 65 aminoacids to about 125 amino acids, about 65 amino acids to about 120 aminoacids, about 65 amino acids to about 115 amino acids, about 65 aminoacids to about 110 amino acids, about 65 amino acids to about 105 aminoacids, about 65 amino acids to about 100 amino acids, about 65 aminoacids to about 95 amino acids, about 65 amino acids to about 90 aminoacids, about 65 amino acids to about 85 amino acids, about 65 aminoacids to about 80 amino acids, about 65 amino acids to about 75 aminoacids, about 65 amino acids to about 70 amino acids, about 70 aminoacids to about 1000 amino acids, about 70 amino acids to about 950 aminoacids, about 70 amino acids to about 900 amino acids, about 70 aminoacids to about 850 amino acids, about 70 amino acids to about 800 aminoacids, about 70 amino acids to about 750 amino acids, about 70 aminoacids to about 700 amino acids, about 70 amino acids to about 650 aminoacids, about 70 amino acids to about 600 amino acids, about 70 aminoacids to about 550 amino acids, about 70 amino acids to about 500 aminoacids, about 70 amino acids to about 450 amino acids, about 70 aminoacids to about 400 amino acids, about 70 amino acids to about 350 aminoacids, about 70 amino acids to about 300 amino acids, about 70 aminoacids to about 280 amino acids, about 70 amino acids to about 260 aminoacids, about 70 amino acids to about 240 amino acids, about 70 aminoacids to about 220 amino acids, about 70 amino acids to about 200 aminoacids, about 70 amino acids to about 195 amino acids, about 70 aminoacids to about 190 amino acids, about 70 amino acids to about 185 aminoacids, about 70 amino acids to about 180 amino acids, about 70 aminoacids to about 175 amino acids, about 70 amino acids to about 170 aminoacids, about 70 amino acids to about 165 amino acids, about 70 aminoacids to about 160 amino acids, about 70 amino acids to about 155 aminoacids, about 70 amino acids to about 150 amino acids, about 70 aminoacids to about 145 amino acids, about 70 amino acids to about 140 aminoacids, about 70 amino acids to about 135 amino acids, about 70 aminoacids to about 130 amino acids, about 70 amino acids to about 125 aminoacids, about 70 amino acids to about 120 amino acids, about 70 aminoacids to about 115 amino acids, about 70 amino acids to about 110 aminoacids, about 70 amino acids to about 105 amino acids, about 70 aminoacids to about 100 amino acids, about 70 amino acids to about 95 aminoacids, about 70 amino acids to about 90 amino acids, about 70 aminoacids to about 85 amino acids, about 70 amino acids to about 80 aminoacids, about 70 amino acids to about 75 amino acids, about 75 aminoacids to about 1000 amino acids, about 75 amino acids to about 950 aminoacids, about 75 amino acids to about 900 amino acids, about 75 aminoacids to about 850 amino acids, about 75 amino acids to about 800 aminoacids, about 75 amino acids to about 750 amino acids, about 75 aminoacids to about 700 amino acids, about 75 amino acids to about 650 aminoacids, about 75 amino acids to about 600 amino acids, about 75 aminoacids to about 550 amino acids, about 75 amino acids to about 500 aminoacids, about 75 amino acids to about 450 amino acids, about 75 aminoacids to about 400 amino acids, about 75 amino acids to about 350 aminoacids, about 75 amino acids to about 300 amino acids, about 75 aminoacids to about 280 amino acids, about 75 amino acids to about 260 aminoacids, about 75 amino acids to about 240 amino acids, about 75 aminoacids to about 220 amino acids, about 75 amino acids to about 200 aminoacids, about 75 amino acids to about 195 amino acids, about 75 aminoacids to about 190 amino acids, about 75 amino acids to about 185 aminoacids, about 75 amino acids to about 180 amino acids, about 75 aminoacids to about 175 amino acids, about 75 amino acids to about 170 aminoacids, about 75 amino acids to about 165 amino acids, about 75 aminoacids to about 160 amino acids, about 75 amino acids to about 155 aminoacids, about 75 amino acids to about 150 amino acids, about 75 aminoacids to about 145 amino acids, about 75 amino acids to about 140 aminoacids, about 75 amino acids to about 135 amino acids, about 75 aminoacids to about 130 amino acids, about 75 amino acids to about 125 aminoacids, about 75 amino acids to about 120 amino acids, about 75 aminoacids to about 115 amino acids, about 75 amino acids to about 110 aminoacids, about 75 amino acids to about 105 amino acids, about 75 aminoacids to about 100 amino acids, about 75 amino acids to about 95 aminoacids, about 75 amino acids to about 90 amino acids, about 75 aminoacids to about 85 amino acids, about 75 amino acids to about 80 aminoacids, about 80 amino acids to about 1000 amino acids, about 80 aminoacids to about 950 amino acids, about 80 amino acids to about 900 aminoacids, about 80 amino acids to about 850 amino acids, about 80 aminoacids to about 800 amino acids, about 80 amino acids to about 750 aminoacids, about 80 amino acids to about 700 amino acids, about 80 aminoacids to about 650 amino acids, about 80 amino acids to about 600 aminoacids, about 80 amino acids to about 550 amino acids, about 80 aminoacids to about 500 amino acids, about 80 amino acids to about 450 aminoacids, about 80 amino acids to about 400 amino acids, about 80 aminoacids to about 350 amino acids, about 80 amino acids to about 300 aminoacids, about 80 amino acids to about 280 amino acids, about 80 aminoacids to about 260 amino acids, about 80 amino acids to about 240 aminoacids, about 80 amino acids to about 220 amino acids, about 80 aminoacids to about 200 amino acids, about 80 amino acids to about 195 aminoacids, about 80 amino acids to about 190 amino acids, about 80 aminoacids to about 185 amino acids, about 80 amino acids to about 180 aminoacids, about 80 amino acids to about 175 amino acids, about 80 aminoacids to about 170 amino acids, about 80 amino acids to about 165 aminoacids, about 80 amino acids to about 160 amino acids, about 80 aminoacids to about 155 amino acids, about 80 amino acids to about 150 aminoacids, about 80 amino acids to about 145 amino acids, about 80 aminoacids to about 140 amino acids, about 80 amino acids to about 135 aminoacids, about 80 amino acids to about 130 amino acids, about 80 aminoacids to about 125 amino acids, about 80 amino acids to about 120 aminoacids, about 80 amino acids to about 115 amino acids, about 80 aminoacids to about 110 amino acids, about 80 amino acids to about 105 aminoacids, about 80 amino acids to about 100 amino acids, about 80 aminoacids to about 95 amino acids, about 80 amino acids to about 90 aminoacids, about 80 amino acids to about 85 amino acids, about 85 aminoacids to about 1000 amino acids, about 85 amino acids to about 950 aminoacids, about 85 amino acids to about 900 amino acids, about 85 aminoacids to about 850 amino acids, about 85 amino acids to about 800 aminoacids, about 85 amino acids to about 750 amino acids, about 85 aminoacids to about 700 amino acids, about 85 amino acids to about 650 aminoacids, about 85 amino acids to about 600 amino acids, about 85 aminoacids to about 550 amino acids, about 85 amino acids to about 500 aminoacids, about 85 amino acids to about 450 amino acids, about 85 aminoacids to about 400 amino acids, about 85 amino acids to about 350 aminoacids, about 85 amino acids to about 300 amino acids, about 85 aminoacids to about 280 amino acids, about 85 amino acids to about 260 aminoacids, about 85 amino acids to about 240 amino acids, about 85 aminoacids to about 220 amino acids, about 85 amino acids to about 200 aminoacids, about 85 amino acids to about 195 amino acids, about 85 aminoacids to about 190 amino acids, about 85 amino acids to about 185 aminoacids, about 85 amino acids to about 180 amino acids, about 85 aminoacids to about 175 amino acids, about 85 amino acids to about 170 aminoacids, about 85 amino acids to about 165 amino acids, about 85 aminoacids to about 160 amino acids, about 85 amino acids to about 155 aminoacids, about 85 amino acids to about 150 amino acids, about 85 aminoacids to about 145 amino acids, about 85 amino acids to about 140 aminoacids, about 85 amino acids to about 135 amino acids, about 85 aminoacids to about 130 amino acids, about 85 amino acids to about 125 aminoacids, about 85 amino acids to about 120 amino acids, about 85 aminoacids to about 115 amino acids, about 85 amino acids to about 110 aminoacids, about 85 amino acids to about 105 amino acids, about 85 aminoacids to about 100 amino acids, about 85 amino acids to about 95 aminoacids, about 85 amino acids to about 90 amino acids, about 90 aminoacids to about 1000 amino acids, about 90 amino acids to about 950 aminoacids, about 90 amino acids to about 900 amino acids, about 90 aminoacids to about 850 amino acids, about 90 amino acids to about 800 aminoacids, about 90 amino acids to about 750 amino acids, about 90 aminoacids to about 700 amino acids, about 90 amino acids to about 650 aminoacids, about 90 amino acids to about 600 amino acids, about 90 aminoacids to about 550 amino acids, about 90 amino acids to about 500 aminoacids, about 90 amino acids to about 450 amino acids, about 90 aminoacids to about 400 amino acids, about 90 amino acids to about 350 aminoacids, about 90 amino acids to about 300 amino acids, about 90 aminoacids to about 280 amino acids, about 90 amino acids to about 260 aminoacids, about 90 amino acids to about 240 amino acids, about 90 aminoacids to about 220 amino acids, about 90 amino acids to about 200 aminoacids, about 90 amino acids to about 195 amino acids, about 90 aminoacids to about 190 amino acids, about 90 amino acids to about 185 aminoacids, about 90 amino acids to about 180 amino acids, about 90 aminoacids to about 175 amino acids, about 90 amino acids to about 170 aminoacids, about 90 amino acids to about 165 amino acids, about 90 aminoacids to about 160 amino acids, about 90 amino acids to about 155 aminoacids, about 90 amino acids to about 150 amino acids, about 90 aminoacids to about 145 amino acids, about 90 amino acids to about 140 aminoacids, about 90 amino acids to about 135 amino acids, about 90 aminoacids to about 130 amino acids, about 90 amino acids to about 125 aminoacids, about 90 amino acids to about 120 amino acids, about 90 aminoacids to about 115 amino acids, about 90 amino acids to about 110 aminoacids, about 90 amino acids to about 105 amino acids, about 90 aminoacids to about 100 amino acids, about 90 amino acids to about 95 aminoacids, about 95 amino acids to about 1000 amino acids, about 95 aminoacids to about 950 amino acids, about 95 amino acids to about 900 aminoacids, about 95 amino acids to about 850 amino acids, about 95 aminoacids to about 800 amino acids, about 95 amino acids to about 750 aminoacids, about 95 amino acids to about 700 amino acids, about 95 aminoacids to about 650 amino acids, about 95 amino acids to about 600 aminoacids, about 95 amino acids to about 550 amino acids, about 95 aminoacids to about 500 amino acids, about 95 amino acids to about 450 aminoacids, about 95 amino acids to about 400 amino acids, about 95 aminoacids to about 350 amino acids, about 95 amino acids to about 300 aminoacids, about 95 amino acids to about 280 amino acids, about 95 aminoacids to about 260 amino acids, about 95 amino acids to about 240 aminoacids, about 95 amino acids to about 220 amino acids, about 95 aminoacids to about 200 amino acids, about 95 amino acids to about 195 aminoacids, about 95 amino acids to about 190 amino acids, about 95 aminoacids to about 185 amino acids, about 95 amino acids to about 180 aminoacids, about 95 amino acids to about 175 amino acids, about 95 aminoacids to about 170 amino acids, about 95 amino acids to about 165 aminoacids, about 95 amino acids to about 160 amino acids, about 95 aminoacids to about 155 amino acids, about 95 amino acids to about 150 aminoacids, about 95 amino acids to about 145 amino acids, about 95 aminoacids to about 140 amino acids, about 95 amino acids to about 135 aminoacids, about 95 amino acids to about 130 amino acids, about 95 aminoacids to about 125 amino acids, about 95 amino acids to about 120 aminoacids, about 95 amino acids to about 115 amino acids, about 95 aminoacids to about 110 amino acids, about 95 amino acids to about 105 aminoacids, about 95 amino acids to about 100 amino acids, about 100 aminoacids to about 1000 amino acids, about 100 amino acids to about 950amino acids, about 100 amino acids to about 900 amino acids, about 100amino acids to about 850 amino acids, about 100 amino acids to about 800amino acids, about 100 amino acids to about 750 amino acids, about 100amino acids to about 700 amino acids, about 100 amino acids to about 650amino acids, about 100 amino acids to about 600 amino acids, about 100amino acids to about 550 amino acids, about 100 amino acids to about 500amino acids, about 100 amino acids to about 450 amino acids, about 100amino acids to about 400 amino acids, about 100 amino acids to about 350amino acids, about 100 amino acids to about 300 amino acids, about 100amino acids to about 280 amino acids, about 100 amino acids to about 260amino acids, about 100 amino acids to about 240 amino acids, about 100amino acids to about 220 amino acids, about 100 amino acids to about 200amino acids, about 100 amino acids to about 195 amino acids, about 100amino acids to about 190 amino acids, about 100 amino acids to about 185amino acids, about 100 amino acids to about 180 amino acids, about 100amino acids to about 175 amino acids, about 100 amino acids to about 170amino acids, about 100 amino acids to about 165 amino acids, about 100amino acids to about 160 amino acids, about 100 amino acids to about 155amino acids, about 100 amino acids to about 150 amino acids, about 100amino acids to about 145 amino acids, about 100 amino acids to about 140amino acids, about 100 amino acids to about 135 amino acids, about 100amino acids to about 130 amino acids, about 100 amino acids to about 125amino acids, about 100 amino acids to about 120 amino acids, about 100amino acids to about 115 amino acids, about 100 amino acids to about 110amino acids, about 100 amino acids to about 105 amino acids, about 105amino acids to about 1000 amino acids, about 105 amino acids to about950 amino acids, about 105 amino acids to about 900 amino acids, about105 amino acids to about 850 amino acids, about 105 amino acids to about800 amino acids, about 105 amino acids to about 750 amino acids, about105 amino acids to about 700 amino acids, about 105 amino acids to about650 amino acids, about 105 amino acids to about 600 amino acids, about105 amino acids to about 550 amino acids, about 105 amino acids to about500 amino acids, about 105 amino acids to about 450 amino acids, about105 amino acids to about 400 amino acids, about 105 amino acids to about350 amino acids, about 105 amino acids to about 300 amino acids, about105 amino acids to about 280 amino acids, about 105 amino acids to about260 amino acids, about 105 amino acids to about 240 amino acids, about105 amino acids to about 220 amino acids, about 105 amino acids to about200 amino acids, about 105 amino acids to about 195 amino acids, about105 amino acids to about 190 amino acids, about 105 amino acids to about185 amino acids, about 105 amino acids to about 180 amino acids, about105 amino acids to about 175 amino acids, about 105 amino acids to about170 amino acids, about 105 amino acids to about 165 amino acids, about105 amino acids to about 160 amino acids, about 105 amino acids to about155 amino acids, about 105 amino acids to about 150 amino acids, about105 amino acids to about 145 amino acids, about 105 amino acids to about140 amino acids, about 105 amino acids to about 135 amino acids, about105 amino acids to about 130 amino acids, about 105 amino acids to about125 amino acids, about 105 amino acids to about 120 amino acids, about105 amino acids to about 115 amino acids, about 105 amino acids to about110 amino acids, about 110 amino acids to about 1000 amino acids, about110 amino acids to about 950 amino acids, about 110 amino acids to about900 amino acids, about 110 amino acids to about 850 amino acids, about110 amino acids to about 800 amino acids, about 110 amino acids to about750 amino acids, about 110 amino acids to about 700 amino acids, about110 amino acids to about 650 amino acids, about 110 amino acids to about600 amino acids, about 110 amino acids to about 550 amino acids, about110 amino acids to about 500 amino acids, about 110 amino acids to about450 amino acids, about 110 amino acids to about 400 amino acids, about110 amino acids to about 350 amino acids, about 110 amino acids to about300 amino acids, about 110 amino acids to about 280 amino acids, about110 amino acids to about 260 amino acids, about 110 amino acids to about240 amino acids, about 110 amino acids to about 220 amino acids, about110 amino acids to about 200 amino acids, about 110 amino acids to about195 amino acids, about 110 amino acids to about 190 amino acids, about110 amino acids to about 185 amino acids, about 110 amino acids to about180 amino acids, about 110 amino acids to about 175 amino acids, about110 amino acids to about 170 amino acids, about 110 amino acids to about165 amino acids, about 110 amino acids to about 160 amino acids, about110 amino acids to about 155 amino acids, about 110 amino acids to about150 amino acids, about 110 amino acids to about 145 amino acids, about110 amino acids to about 140 amino acids, about 110 amino acids to about135 amino acids, about 110 amino acids to about 130 amino acids, about110 amino acids to about 125 amino acids, about 110 amino acids to about120 amino acids, about 110 amino acids to about 115 amino acids, about115 amino acids to about 1000 amino acids, about 115 amino acids toabout 950 amino acids, about 115 amino acids to about 900 amino acids,about 115 amino acids to about 850 amino acids, about 115 amino acids toabout 800 amino acids, about 115 amino acids to about 750 amino acids,about 115 amino acids to about 700 amino acids, about 115 amino acids toabout 650 amino acids, about 115 amino acids to about 600 amino acids,about 115 amino acids to about 550 amino acids, about 115 amino acids toabout 500 amino acids, about 115 amino acids to about 450 amino acids,about 115 amino acids to about 400 amino acids, about 115 amino acids toabout 350 amino acids, about 115 amino acids to about 300 amino acids,about 115 amino acids to about 280 amino acids, about 115 amino acids toabout 260 amino acids, about 115 amino acids to about 240 amino acids,about 115 amino acids to about 220 amino acids, about 115 amino acids toabout 200 amino acids, about 115 amino acids to about 195 amino acids,about 115 amino acids to about 190 amino acids, about 115 amino acids toabout 185 amino acids, about 115 amino acids to about 180 amino acids,about 115 amino acids to about 175 amino acids, about 115 amino acids toabout 170 amino acids, about 115 amino acids to about 165 amino acids,about 115 amino acids to about 160 amino acids, about 115 amino acids toabout 155 amino acids, about 115 amino acids to about 150 amino acids,about 115 amino acids to about 145 amino acids, about 115 amino acids toabout 140 amino acids, about 115 amino acids to about 135 amino acids,about 115 amino acids to about 130 amino acids, about 115 amino acids toabout 125 amino acids, about 115 amino acids to about 120 amino acids,about 120 amino acids to about 1000 amino acids, about 120 amino acidsto about 950 amino acids, about 120 amino acids to about 900 aminoacids, about 120 amino acids to about 850 amino acids, about 120 aminoacids to about 800 amino acids, about 120 amino acids to about 750 aminoacids, about 120 amino acids to about 700 amino acids, about 120 aminoacids to about 650 amino acids, about 120 amino acids to about 600 aminoacids, about 120 amino acids to about 550 amino acids, about 120 aminoacids to about 500 amino acids, about 120 amino acids to about 450 aminoacids, about 120 amino acids to about 400 amino acids, about 120 aminoacids to about 350 amino acids, about 120 amino acids to about 300 aminoacids, about 120 amino acids to about 280 amino acids, about 120 aminoacids to about 260 amino acids, about 120 amino acids to about 240 aminoacids, about 120 amino acids to about 220 amino acids, about 120 aminoacids to about 200 amino acids, about 120 amino acids to about 195 aminoacids, about 120 amino acids to about 190 amino acids, about 120 aminoacids to about 185 amino acids, about 120 amino acids to about 180 aminoacids, about 120 amino acids to about 175 amino acids, about 120 aminoacids to about 170 amino acids, about 120 amino acids to about 165 aminoacids, about 120 amino acids to about 160 amino acids, about 120 aminoacids to about 155 amino acids, about 120 amino acids to about 150 aminoacids, about 120 amino acids to about 145 amino acids, about 120 aminoacids to about 140 amino acids, about 120 amino acids to about 135 aminoacids, about 120 amino acids to about 130 amino acids, about 120 aminoacids to about 125 amino acids, about 125 amino acids to about 1000amino acids, about 125 amino acids to about 950 amino acids, about 125amino acids to about 900 amino acids, about 125 amino acids to about 850amino acids, about 125 amino acids to about 800 amino acids, about 125amino acids to about 750 amino acids, about 125 amino acids to about 700amino acids, about 125 amino acids to about 650 amino acids, about 125amino acids to about 600 amino acids, about 125 amino acids to about 550amino acids, about 125 amino acids to about 500 amino acids, about 125amino acids to about 450 amino acids, about 125 amino acids to about 400amino acids, about 125 amino acids to about 350 amino acids, about 125amino acids to about 300 amino acids, about 125 amino acids to about 280amino acids, about 125 amino acids to about 260 amino acids, about 125amino acids to about 240 amino acids, about 125 amino acids to about 220amino acids, about 125 amino acids to about 200 amino acids, about 125amino acids to about 195 amino acids, about 125 amino acids to about 190amino acids, about 125 amino acids to about 185 amino acids, about 125amino acids to about 180 amino acids, about 125 amino acids to about 175amino acids, about 125 amino acids to about 170 amino acids, about 125amino acids to about 165 amino acids, about 125 amino acids to about 160amino acids, about 125 amino acids to about 155 amino acids, about 125amino acids to about 150 amino acids, about 125 amino acids to about 145amino acids, about 125 amino acids to about 140 amino acids, about 125amino acids to about 135 amino acids, about 125 amino acids to about 130amino acids, about 130 amino acids to about 1000 amino acids, about 130amino acids to about 950 amino acids, about 130 amino acids to about 900amino acids, about 130 amino acids to about 850 amino acids, about 130amino acids to about 800 amino acids, about 130 amino acids to about 750amino acids, about 130 amino acids to about 700 amino acids, about 130amino acids to about 650 amino acids, about 130 amino acids to about 600amino acids, about 130 amino acids to about 550 amino acids, about 130amino acids to about 500 amino acids, about 130 amino acids to about 450amino acids, about 130 amino acids to about 400 amino acids, about 130amino acids to about 350 amino acids, about 130 amino acids to about 300amino acids, about 130 amino acids to about 280 amino acids, about 130amino acids to about 260 amino acids, about 130 amino acids to about 240amino acids, about 130 amino acids to about 220 amino acids, about 130amino acids to about 200 amino acids, about 130 amino acids to about 195amino acids, about 130 amino acids to about 190 amino acids, about 130amino acids to about 185 amino acids, about 130 amino acids to about 180amino acids, about 130 amino acids to about 175 amino acids, about 130amino acids to about 170 amino acids, about 130 amino acids to about 165amino acids, about 130 amino acids to about 160 amino acids, about 130amino acids to about 155 amino acids, about 130 amino acids to about 150amino acids, about 130 amino acids to about 145 amino acids, about 130amino acids to about 140 amino acids, about 130 amino acids to about 135amino acids, about 135 amino acids to about 1000 amino acids, about 135amino acids to about 950 amino acids, about 135 amino acids to about 900amino acids, about 135 amino acids to about 850 amino acids, about 135amino acids to about 800 amino acids, about 135 amino acids to about 750amino acids, about 135 amino acids to about 700 amino acids, about 135amino acids to about 650 amino acids, about 135 amino acids to about 600amino acids, about 135 amino acids to about 550 amino acids, about 135amino acids to about 500 amino acids, about 135 amino acids to about 450amino acids, about 135 amino acids to about 400 amino acids, about 135amino acids to about 350 amino acids, about 135 amino acids to about 300amino acids, about 135 amino acids to about 280 amino acids, about 135amino acids to about 260 amino acids, about 135 amino acids to about 240amino acids, about 135 amino acids to about 220 amino acids, about 135amino acids to about 200 amino acids, about 135 amino acids to about 195amino acids, about 135 amino acids to about 190 amino acids, about 135amino acids to about 185 amino acids, about 135 amino acids to about 180amino acids, about 135 amino acids to about 175 amino acids, about 135amino acids to about 170 amino acids, about 135 amino acids to about 165amino acids, about 135 amino acids to about 160 amino acids, about 135amino acids to about 155 amino acids, about 135 amino acids to about 150amino acids, about 135 amino acids to about 145 amino acids, about 135amino acids to about 140 amino acids, about 140 amino acids to about1000 amino acids, about 140 amino acids to about 950 amino acids, about140 amino acids to about 900 amino acids, about 140 amino acids to about850 amino acids, about 140 amino acids to about 800 amino acids, about140 amino acids to about 750 amino acids, about 140 amino acids to about700 amino acids, about 140 amino acids to about 650 amino acids, about140 amino acids to about 600 amino acids, about 140 amino acids to about550 amino acids, about 140 amino acids to about 500 amino acids, about140 amino acids to about 450 amino acids, about 140 amino acids to about400 amino acids, about 140 amino acids to about 350 amino acids, about140 amino acids to about 300 amino acids, about 140 amino acids to about280 amino acids, about 140 amino acids to about 260 amino acids, about140 amino acids to about 240 amino acids, about 140 amino acids to about220 amino acids, about 140 amino acids to about 200 amino acids, about140 amino acids to about 195 amino acids, about 140 amino acids to about190 amino acids, about 140 amino acids to about 185 amino acids, about140 amino acids to about 180 amino acids, about 140 amino acids to about175 amino acids, about 140 amino acids to about 170 amino acids, about140 amino acids to about 165 amino acids, about 140 amino acids to about160 amino acids, about 140 amino acids to about 155 amino acids, about140 amino acids to about 150 amino acids, about 140 amino acids to about145 amino acids, about 145 amino acids to about 1000 amino acids, about145 amino acids to about 950 amino acids, about 145 amino acids to about900 amino acids, about 145 amino acids to about 850 amino acids, about145 amino acids to about 800 amino acids, about 145 amino acids to about750 amino acids, about 145 amino acids to about 700 amino acids, about145 amino acids to about 650 amino acids, about 145 amino acids to about600 amino acids, about 145 amino acids to about 550 amino acids, about145 amino acids to about 500 amino acids, about 145 amino acids to about450 amino acids, about 145 amino acids to about 400 amino acids, about145 amino acids to about 350 amino acids, about 145 amino acids to about300 amino acids, about 145 amino acids to about 280 amino acids, about145 amino acids to about 260 amino acids, about 145 amino acids to about240 amino acids, about 145 amino acids to about 220 amino acids, about145 amino acids to about 200 amino acids, about 145 amino acids to about195 amino acids, about 145 amino acids to about 190 amino acids, about145 amino acids to about 185 amino acids, about 145 amino acids to about180 amino acids, about 145 amino acids to about 175 amino acids, about145 amino acids to about 170 amino acids, about 145 amino acids to about165 amino acids, about 145 amino acids to about 160 amino acids, about145 amino acids to about 155 amino acids, about 145 amino acids to about150 amino acids, about 150 amino acids to about 1000 amino acids, about150 amino acids to about 950 amino acids, about 150 amino acids to about900 amino acids, about 150 amino acids to about 850 amino acids, about150 amino acids to about 800 amino acids, about 150 amino acids to about750 amino acids, about 150 amino acids to about 700 amino acids, about150 amino acids to about 650 amino acids, about 150 amino acids to about600 amino acids, about 150 amino acids to about 550 amino acids, about150 amino acids to about 500 amino acids, about 150 amino acids to about450 amino acids, about 150 amino acids to about 400 amino acids, about150 amino acids to about 350 amino acids, about 150 amino acids to about300 amino acids, about 150 amino acids to about 280 amino acids, about150 amino acids to about 260 amino acids, about 150 amino acids to about240 amino acids, about 150 amino acids to about 220 amino acids, about150 amino acids to about 200 amino acids, about 150 amino acids to about195 amino acids, about 150 amino acids to about 190 amino acids, about150 amino acids to about 185 amino acids, about 150 amino acids to about180 amino acids, about 150 amino acids to about 175 amino acids, about150 amino acids to about 170 amino acids, about 150 amino acids to about165 amino acids, about 150 amino acids to about 160 amino acids, about150 amino acids to about 155 amino acids, about 155 amino acids to about1000 amino acids, about 155 amino acids to about 950 amino acids, about155 amino acids to about 900 amino acids, about 155 amino acids to about850 amino acids, about 155 amino acids to about 800 amino acids, about155 amino acids to about 750 amino acids, about 155 amino acids to about700 amino acids, about 155 amino acids to about 650 amino acids, about155 amino acids to about 600 amino acids, about 155 amino acids to about550 amino acids, about 155 amino acids to about 500 amino acids, about155 amino acids to about 450 amino acids, about 155 amino acids to about400 amino acids, about 155 amino acids to about 350 amino acids, about155 amino acids to about 300 amino acids, about 155 amino acids to about280 amino acids, about 155 amino acids to about 260 amino acids, about155 amino acids to about 240 amino acids, about 155 amino acids to about220 amino acids, about 155 amino acids to about 200 amino acids, about155 amino acids to about 195 amino acids, about 155 amino acids to about190 amino acids, about 155 amino acids to about 185 amino acids, about155 amino acids to about 180 amino acids, about 155 amino acids to about175 amino acids, about 155 amino acids to about 170 amino acids, about155 amino acids to about 165 amino acids, about 155 amino acids to about160 amino acids, about 160 amino acids to about 1000 amino acids, about160 amino acids to about 950 amino acids, about 160 amino acids to about900 amino acids, about 160 amino acids to about 850 amino acids, about160 amino acids to about 800 amino acids, about 160 amino acids to about750 amino acids, about 160 amino acids to about 700 amino acids, about160 amino acids to about 650 amino acids, about 160 amino acids to about600 amino acids, about 160 amino acids to about 550 amino acids, about160 amino acids to about 500 amino acids, about 160 amino acids to about450 amino acids, about 160 amino acids to about 400 amino acids, about160 amino acids to about 350 amino acids, about 160 amino acids to about300 amino acids, about 160 amino acids to about 280 amino acids, about160 amino acids to about 260 amino acids, about 160 amino acids to about240 amino acids, about 160 amino acids to about 220 amino acids, about160 amino acids to about 200 amino acids, about 160 amino acids to about195 amino acids, about 160 amino acids to about 190 amino acids, about160 amino acids to about 185 amino acids, about 160 amino acids to about180 amino acids, about 160 amino acids to about 175 amino acids, about160 amino acids to about 170 amino acids, about 160 amino acids to about165 amino acids, about 165 amino acids to about 1000 amino acids, about165 amino acids to about 950 amino acids, about 165 amino acids to about900 amino acids, about 165 amino acids to about 850 amino acids, about165 amino acids to about 800 amino acids, about 165 amino acids to about750 amino acids, about 165 amino acids to about 700 amino acids, about165 amino acids to about 650 amino acids, about 165 amino acids to about600 amino acids, about 165 amino acids to about 550 amino acids, about165 amino acids to about 500 amino acids, about 165 amino acids to about450 amino acids, about 165 amino acids to about 400 amino acids, about165 amino acids to about 350 amino acids, about 165 amino acids to about300 amino acids, about 165 amino acids to about 280 amino acids, about165 amino acids to about 260 amino acids, about 165 amino acids to about240 amino acids, about 165 amino acids to about 220 amino acids, about165 amino acids to about 200 amino acids, about 165 amino acids to about195 amino acids, about 165 amino acids to about 190 amino acids, about165 amino acids to about 185 amino acids, about 165 amino acids to about180 amino acids, about 165 amino acids to about 175 amino acids, about165 amino acids to about 170 amino acids, about 170 amino acids to about1000 amino acids, about 170 amino acids to about 950 amino acids, about170 amino acids to about 900 amino acids, about 170 amino acids to about850 amino acids, about 170 amino acids to about 800 amino acids, about170 amino acids to about 750 amino acids, about 170 amino acids to about700 amino acids, about 170 amino acids to about 650 amino acids, about170 amino acids to about 600 amino acids, about 170 amino acids to about550 amino acids, about 170 amino acids to about 500 amino acids, about170 amino acids to about 450 amino acids, about 170 amino acids to about400 amino acids, about 170 amino acids to about 350 amino acids, about170 amino acids to about 300 amino acids, about 170 amino acids to about280 amino acids, about 170 amino acids to about 260 amino acids, about170 amino acids to about 240 amino acids, about 170 amino acids to about220 amino acids, about 170 amino acids to about 200 amino acids, about170 amino acids to about 195 amino acids, about 170 amino acids to about190 amino acids, about 170 amino acids to about 185 amino acids, about170 amino acids to about 180 amino acids, about 170 amino acids to about175 amino acids, about 175 amino acids to about 1000 amino acids, about175 amino acids to about 950 amino acids, about 175 amino acids to about900 amino acids, about 175 amino acids to about 850 amino acids, about175 amino acids to about 800 amino acids, about 175 amino acids to about750 amino acids, about 175 amino acids to about 700 amino acids, about175 amino acids to about 650 amino acids, about 175 amino acids to about600 amino acids, about 175 amino acids to about 550 amino acids, about175 amino acids to about 500 amino acids, about 175 amino acids to about450 amino acids, about 175 amino acids to about 400 amino acids, about175 amino acids to about 350 amino acids, about 175 amino acids to about300 amino acids, about 175 amino acids to about 280 amino acids, about175 amino acids to about 260 amino acids, about 175 amino acids to about240 amino acids, about 175 amino acids to about 220 amino acids, about175 amino acids to about 200 amino acids, about 175 amino acids to about195 amino acids, about 175 amino acids to about 190 amino acids, about175 amino acids to about 185 amino acids, about 175 amino acids to about180 amino acids, about 180 amino acids to about 1000 amino acids, about180 amino acids to about 950 amino acids, about 180 amino acids to about900 amino acids, about 180 amino acids to about 850 amino acids, about180 amino acids to about 800 amino acids, about 180 amino acids to about750 amino acids, about 180 amino acids to about 700 amino acids, about180 amino acids to about 650 amino acids, about 180 amino acids to about600 amino acids, about 180 amino acids to about 550 amino acids, about180 amino acids to about 500 amino acids, about 180 amino acids to about450 amino acids, about 180 amino acids to about 400 amino acids, about180 amino acids to about 350 amino acids, about 180 amino acids to about300 amino acids, about 180 amino acids to about 280 amino acids, about180 amino acids to about 260 amino acids, about 180 amino acids to about240 amino acids, about 180 amino acids to about 220 amino acids, about180 amino acids to about 200 amino acids, about 180 amino acids to about195 amino acids, about 180 amino acids to about 190 amino acids, about180 amino acids to about 185 amino acids, about 185 amino acids to about1000 amino acids, about 185 amino acids to about 950 amino acids, about185 amino acids to about 900 amino acids, about 185 amino acids to about850 amino acids, about 185 amino acids to about 800 amino acids, about185 amino acids to about 750 amino acids, about 185 amino acids to about700 amino acids, about 185 amino acids to about 650 amino acids, about185 amino acids to about 600 amino acids, about 185 amino acids to about550 amino acids, about 185 amino acids to about 500 amino acids, about185 amino acids to about 450 amino acids, about 185 amino acids to about400 amino acids, about 185 amino acids to about 350 amino acids, about185 amino acids to about 300 amino acids, about 185 amino acids to about280 amino acids, about 185 amino acids to about 260 amino acids, about185 amino acids to about 240 amino acids, about 185 amino acids to about220 amino acids, about 185 amino acids to about 200 amino acids, about185 amino acids to about 195 amino acids, about 185 amino acids to about190 amino acids, about 190 amino acids to about 1000 amino acids, about190 amino acids to about 950 amino acids, about 190 amino acids to about900 amino acids, about 190 amino acids to about 850 amino acids, about190 amino acids to about 800 amino acids, about 190 amino acids to about750 amino acids, about 190 amino acids to about 700 amino acids, about190 amino acids to about 650 amino acids, about 190 amino acids to about600 amino acids, about 190 amino acids to about 550 amino acids, about190 amino acids to about 500 amino acids, about 190 amino acids to about450 amino acids, about 190 amino acids to about 400 amino acids, about190 amino acids to about 350 amino acids, about 190 amino acids to about300 amino acids, about 190 amino acids to about 280 amino acids, about190 amino acids to about 260 amino acids, about 190 amino acids to about240 amino acids, about 190 amino acids to about 220 amino acids, about190 amino acids to about 200 amino acids, about 190 amino acids to about195 amino acids, about 195 amino acids to about 1000 amino acids, about195 amino acids to about 950 amino acids, about 195 amino acids to about900 amino acids, about 195 amino acids to about 850 amino acids, about195 amino acids to about 800 amino acids, about 195 amino acids to about750 amino acids, about 195 amino acids to about 700 amino acids, about195 amino acids to about 650 amino acids, about 195 amino acids to about600 amino acids, about 195 amino acids to about 550 amino acids, about195 amino acids to about 500 amino acids, about 195 amino acids to about450 amino acids, about 195 amino acids to about 400 amino acids, about195 amino acids to about 350 amino acids, about 195 amino acids to about300 amino acids, about 195 amino acids to about 280 amino acids, about195 amino acids to about 260 amino acids, about 195 amino acids to about240 amino acids, about 195 amino acids to about 220 amino acids, about195 amino acids to about 200 amino acids, about 200 amino acids to about1000 amino acids, about 200 amino acids to about 950 amino acids, about200 amino acids to about 900 amino acids, about 200 amino acids to about850 amino acids, about 200 amino acids to about 800 amino acids, about200 amino acids to about 750 amino acids, about 200 amino acids to about700 amino acids, about 200 amino acids to about 650 amino acids, about200 amino acids to about 600 amino acids, about 200 amino acids to about550 amino acids, about 200 amino acids to about 500 amino acids, about200 amino acids to about 450 amino acids, about 200 amino acids to about400 amino acids, about 200 amino acids to about 350 amino acids, about200 amino acids to about 300 amino acids, about 200 amino acids to about280 amino acids, about 200 amino acids to about 260 amino acids, about200 amino acids to about 240 amino acids, about 200 amino acids to about220 amino acids, about 220 amino acids to about 1000 amino acids, about220 amino acids to about 950 amino acids, about 220 amino acids to about900 amino acids, about 220 amino acids to about 850 amino acids, about220 amino acids to about 800 amino acids, about 220 amino acids to about750 amino acids, about 220 amino acids to about 700 amino acids, about220 amino acids to about 650 amino acids, about 220 amino acids to about600 amino acids, about 220 amino acids to about 550 amino acids, about220 amino acids to about 500 amino acids, about 220 amino acids to about450 amino acids, about 220 amino acids to about 400 amino acids, about220 amino acids to about 350 amino acids, about 220 amino acids to about300 amino acids, about 220 amino acids to about 280 amino acids, about220 amino acids to about 260 amino acids, about 220 amino acids to about240 amino acids, about 240 amino acids to about 1000 amino acids, about240 amino acids to about 950 amino acids, about 240 amino acids to about900 amino acids, about 240 amino acids to about 850 amino acids, about240 amino acids to about 800 amino acids, about 240 amino acids to about750 amino acids, about 240 amino acids to about 700 amino acids, about240 amino acids to about 650 amino acids, about 240 amino acids to about600 amino acids, about 240 amino acids to about 550 amino acids, about240 amino acids to about 500 amino acids, about 240 amino acids to about450 amino acids, about 240 amino acids to about 400 amino acids, about240 amino acids to about 350 amino acids, about 240 amino acids to about300 amino acids, about 240 amino acids to about 280 amino acids, about240 amino acids to about 260 amino acids, about 260 amino acids to about1000 amino acids, about 260 amino acids to about 950 amino acids, about260 amino acids to about 900 amino acids, about 260 amino acids to about850 amino acids, about 260 amino acids to about 800 amino acids, about260 amino acids to about 750 amino acids, about 260 amino acids to about700 amino acids, about 260 amino acids to about 650 amino acids, about260 amino acids to about 600 amino acids, about 260 amino acids to about550 amino acids, about 260 amino acids to about 500 amino acids, about260 amino acids to about 450 amino acids, about 260 amino acids to about400 amino acids, about 260 amino acids to about 350 amino acids, about260 amino acids to about 300 amino acids, about 260 amino acids to about280 amino acids, about 280 amino acids to about 1000 amino acids, about280 amino acids to about 950 amino acids, about 280 amino acids to about900 amino acids, about 280 amino acids to about 850 amino acids, about280 amino acids to about 800 amino acids, about 280 amino acids to about750 amino acids, about 280 amino acids to about 700 amino acids, about280 amino acids to about 650 amino acids, about 280 amino acids to about600 amino acids, about 280 amino acids to about 550 amino acids, about280 amino acids to about 500 amino acids, about 280 amino acids to about450 amino acids, about 280 amino acids to about 400 amino acids, about280 amino acids to about 350 amino acids, about 280 amino acids to about300 amino acids, about 300 amino acids to about 1000 amino acids, about300 amino acids to about 950 amino acids, about 300 amino acids to about900 amino acids, about 300 amino acids to about 850 amino acids, about300 amino acids to about 800 amino acids, about 300 amino acids to about750 amino acids, about 300 amino acids to about 700 amino acids, about300 amino acids to about 650 amino acids, about 300 amino acids to about600 amino acids, about 300 amino acids to about 550 amino acids, about300 amino acids to about 500 amino acids, about 300 amino acids to about450 amino acids, about 300 amino acids to about 400 amino acids, about300 amino acids to about 350 amino acids, about 350 amino acids to about1000 amino acids, about 350 amino acids to about 950 amino acids, about350 amino acids to about 900 amino acids, about 350 amino acids to about850 amino acids, about 350 amino acids to about 800 amino acids, about350 amino acids to about 750 amino acids, about 350 amino acids to about700 amino acids, about 350 amino acids to about 650 amino acids, about350 amino acids to about 600 amino acids, about 350 amino acids to about550 amino acids, about 350 amino acids to about 500 amino acids, about350 amino acids to about 450 amino acids, about 350 amino acids to about400 amino acids, about 400 amino acids to about 1000 amino acids, about400 amino acids to about 950 amino acids, about 400 amino acids to about900 amino acids, about 400 amino acids to about 850 amino acids, about400 amino acids to about 800 amino acids, about 400 amino acids to about750 amino acids, about 400 amino acids to about 700 amino acids, about400 amino acids to about 650 amino acids, about 400 amino acids to about600 amino acids, about 400 amino acids to about 550 amino acids, about400 amino acids to about 500 amino acids, about 400 amino acids to about450 amino acids, about 450 amino acids to about 1000 amino acids, about450 amino acids to about 950 amino acids, about 450 amino acids to about900 amino acids, about 450 amino acids to about 850 amino acids, about450 amino acids to about 800 amino acids, about 450 amino acids to about750 amino acids, about 450 amino acids to about 700 amino acids, about450 amino acids to about 650 amino acids, about 450 amino acids to about600 amino acids, about 450 amino acids to about 550 amino acids, about450 amino acids to about 500 amino acids, about 500 amino acids to about1000 amino acids, about 500 amino acids to about 950 amino acids, about500 amino acids to about 900 amino acids, about 500 amino acids to about850 amino acids, about 500 amino acids to about 800 amino acids, about500 amino acids to about 750 amino acids, about 500 amino acids to about700 amino acids, about 500 amino acids to about 650 amino acids, about500 amino acids to about 600 amino acids, about 500 amino acids to about550 amino acids, about 550 amino acids to about 1000 amino acids, about550 amino acids to about 950 amino acids, about 550 amino acids to about900 amino acids, about 550 amino acids to about 850 amino acids, about550 amino acids to about 800 amino acids, about 550 amino acids to about750 amino acids, about 550 amino acids to about 700 amino acids, about550 amino acids to about 650 amino acids, about 550 amino acids to about600 amino acids, about 600 amino acids to about 1000 amino acids, about600 amino acids to about 950 amino acids, about 600 amino acids to about900 amino acids, about 600 amino acids to about 850 amino acids, about600 amino acids to about 800 amino acids, about 600 amino acids to about750 amino acids, about 600 amino acids to about 700 amino acids, about600 amino acids to about 650 amino acids, about 650 amino acids to about1000 amino acids, about 650 amino acids to about 950 amino acids, about650 amino acids to about 900 amino acids, about 650 amino acids to about850 amino acids, about 650 amino acids to about 800 amino acids, about650 amino acids to about 750 amino acids, about 650 amino acids to about700 amino acids, about 700 amino acids to about 1000 amino acids, about700 amino acids to about 950 amino acids, about 700 amino acids to about900 amino acids, about 700 amino acids to about 850 amino acids, about700 amino acids to about 800 amino acids, about 700 amino acids to about750 amino acids, about 750 amino acids to about 1000 amino acids, about750 amino acids to about 950 amino acids, about 750 amino acids to about900 amino acids, about 750 amino acids to about 850 amino acids, about750 amino acids to about 800 amino acids, about 800 amino acids to about1000 amino acids, about 800 amino acids to about 950 amino acids, about800 amino acids to about 900 amino acids, about 800 amino acids to about850 amino acids, about 850 amino acids to about 1000 amino acids, about850 amino acids to about 950 amino acids, about 850 amino acids to about900 amino acids, about 900 amino acids to about 1000 amino acids, about900 amino acids to about 950 amino acids, or about 950 amino acids toabout 1000 amino acids.

Any of the target-binding domains described herein can bind to itstarget with a dissociation equilibrium constant (K_(D)) of less than1×10⁻⁷ M, less than 1×10⁻⁸ M, less than 1×10⁻⁹ M, less than 1×10⁻¹⁰ Mless than 1×10⁻¹¹ M, less than 1×10⁻¹² M, or less than 1×10⁻¹³ M. Insome embodiments, the antigen-binding protein construct provided hereincan bind to an identifying antigen with a K_(D) of about 1×10⁻³ M toabout 1×10⁻⁵ M, about 1×10⁻⁴ M to about 1×10⁻⁸ M, about 1×10⁻⁵ M toabout 1×10⁻⁷ M, about 1×10⁻⁶ M to about 1×10⁻⁸ M, about 1×10⁻⁷ M toabout 1×10⁻⁹ M, about 1×10⁻⁸ M to about 1×10⁻¹⁰ M, or about 1×10⁻⁹ M toabout 1×10⁻¹¹ M (inclusive).

Any of the target-binding domains described herein can bind to itstarget with a K_(D) of between about 1 pM to about 30 nM (e.g., about 1pM to about 25 nM, about 1 pM to about 20 nM, about 1 pM to about 15 nM,about 1 pM to about 10 nM, about 1 pM to about 5 nM, about 1 pM to about2 nM, about 1 pM to about 1 nM, about 1 pM to about 950 pM, about 1 pMto about 900 pM, about 1 pM to about 850 pM, about 1 pM to about 800 pM,about 1 pM to about 750 pM, about 1 pM to about 700 pM, about 1 pM toabout 650 pM, about 1 pM to about 600 pM, about 1 pM to about 550 pM,about 1 pM to about 500 pM, about 1 pM to about 450 pM, about 1 pM toabout 400 pM, about 1 pM to about 350 pM, about 1 pM to about 300 pM,about 1 pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM toabout 150 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM,about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM toabout 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 1 pM toabout 3 pM, about 1 pM to about 2 pM, about 2 pM to about 30 nM, about 2pM to about 25 nM, about 2 pM to about 20 nM, about 2 pM to about 15 nM,about 2 pM to about 10 nM, about 2 pM to about 5 nM, about 2 pM to about2 nM, about 2 pM to about 1 nM, about 2 pM to about 950 pM, about 2 pMto about 900 pM, about 2 pM to about 850 pM, about 2 pM to about 800 pM,about 2 pM to about 750 pM, about 2 pM to about 700 pM, about 2 pM toabout 650 pM, about 2 pM to about 600 pM, about 2 pM to about 550 pM,about 2 pM to about 500 pM, about 2 pM to about 450 pM, about 2 pM toabout 400 pM, about 2 pM to about 350 pM, about 2 pM to about 300 pM,about 2 pM to about 250 pM, about 2 pM to about 200 pM, about 2 pM toabout 150 pM, about 2 pM to about 100 pM, about 2 pM to about 90 pM,about 2 pM to about 80 pM, about 2 pM to about 70 pM, about 2 pM toabout 60 pM, about 2 pM to about 50 pM, about 2 pM to about 40 pM, about2 pM to about 30 pM, about 2 pM to about 20 pM, about 2 pM to about 10pM, about 2 pM to about 5 pM, about 2 pM to about 4 pM, about 2 pM toabout 3 pM, about 5 pM to about 30 nM, about 5 pM to about 25 nM, about5 pM to about 20 nM, about 5 pM to about 15 nM, about 5 pM to about 10nM, about 5 pM to about 5 nM, about 5 pM to about 2 nM, about 5 pM toabout 1 nM, about 5 pM to about 950 pM, about 5 pM to about 900 pM,about 5 pM to about 850 pM, about 5 pM to about 800 pM, about 5 pM toabout 750 pM, about 5 pM to about 700 pM, about 5 pM to about 650 pM,about 5 pM to about 600 pM, about 5 pM to about 550 pM, about 5 pM toabout 500 pM, about 5 pM to about 450 pM, about 5 pM to about 400 pM,about 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM toabout 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM,about 5 pM to about 100 pM, about 5 pM to about 90 pM, about 5 pM toabout 80 pM, about 5 pM to about 70 pM, about 5 pM to about 60 pM, about5 pM to about 50 pM, about 5 pM to about 40 pM, about 5 pM to about 30pM, about 5 pM to about 20 pM, about 5 pM to about 10 pM, about 10 pM toabout 30 nM, about 10 pM to about 25 nM, about 10 pM to about 20 nM,about 10 pM to about 15 nM, about 10 pM to about 10 nM, about 10 pM toabout 5 nM, about 10 pM to about 2 nM, about 10 pM to about 1 nM, about10 pM to about 950 pM, about 10 pM to about 900 pM, about 10 pM to about850 pM, about 10 pM to about 800 pM, about 10 pM to about 750 pM, about10 pM to about 700 pM, about 10 pM to about 650 pM, about 10 pM to about600 pM, about 10 pM to about 550 pM, about 10 pM to about 500 pM, about10 pM to about 450 pM, about 10 pM to about 400 pM, about 10 pM to about350 pM, about 10 pM to about 300 pM, about 10 pM to about 250 pM, about10 pM to about 200 pM, about 10 pM to about 150 pM, about 10 pM to about100 pM, about 10 pM to about 90 pM, about 10 pM to about 80 pM, about 10pM to about 70 pM, about 10 pM to about 60 pM, about 10 pM to about 50pM, about 10 pM to about 40 pM, about 10 pM to about 30 pM, about 10 pMto about 20 pM, about 15 pM to about 30 nM, about 15 pM to about 25 nM,about 15 pM to about 20 nM, about 15 pM to about 15 nM, about 15 pM toabout 10 nM, about 15 pM to about 5 nM, about 15 pM to about 2 nM, about15 pM to about 1 nM, about 15 pM to about 950 pM, about 15 pM to about900 pM, about 15 pM to about 850 pM, about 15 pM to about 800 pM, about15 pM to about 750 pM, about 15 pM to about 700 pM, about 15 pM to about650 pM, about 15 pM to about 600 pM, about 15 pM to about 550 pM, about15 pM to about 500 pM, about 15 pM to about 450 pM, about 15 pM to about400 pM, about 15 pM to about 350 pM, about 15 pM to about 300 pM, about15 pM to about 250 pM, about 15 pM to about 200 pM, about 15 pM to about150 pM, about 15 pM to about 100 pM, about 15 pM to about 90 pM, about15 pM to about 80 pM, about 15 pM to about 70 pM, about 15 pM to about60 pM, about 15 pM to about 50 pM, about 15 pM to about 40 pM, about 15pM to about 30 pM, about 15 pM to about 20 pM, about 20 pM to about 30nM, about 20 pM to about 25 nM, about 20 pM to about 20 nM, about 20 pMto about 15 nM, about 20 pM to about 10 nM, about 20 pM to about 5 nM,about 20 pM to about 2 nM, about 20 pM to about 1 nM, about 20 pM toabout 950 pM, about 20 pM to about 900 pM, about 20 pM to about 850 pM,about 20 pM to about 800 pM, about 20 pM to about 750 pM, about 20 pM toabout 700 pM, about 20 pM to about 650 pM, about 20 pM to about 600 pM,about 20 pM to about 550 pM, about 20 pM to about 500 pM, about 20 pM toabout 450 pM, about 20 pM to about 400 pM, about 20 pM to about 350 pM,about 20 pM to about 300 pM, about 20 pM to about 250 pM, about 20 pM toabout 20 pM, about 200 pM to about 150 pM, about 20 pM to about 100 pM,about 20 pM to about 90 pM, about 20 pM to about 80 pM, about 20 pM toabout 70 pM, about 20 pM to about 60 pM, about 20 pM to about 50 pM,about 20 pM to about 40 pM, about 20 pM to about 30 pM, about 30 pM toabout 30 nM, about 30 pM to about 25 nM, about 30 pM to about 30 nM,about 30 pM to about 15 nM, about 30 pM to about 10 nM, about 30 pM toabout 5 nM, about 30 pM to about 2 nM, about 30 pM to about 1 nM, about30 pM to about 950 pM, about 30 pM to about 900 pM, about 30 pM to about850 pM, about 30 pM to about 800 pM, about 30 pM to about 750 pM, about30 pM to about 700 pM, about 30 pM to about 650 pM, about 30 pM to about600 pM, about 30 pM to about 550 pM, about 30 pM to about 500 pM, about30 pM to about 450 pM, about 30 pM to about 400 pM, about 30 pM to about350 pM, about 30 pM to about 300 pM, about 30 pM to about 250 pM, about30 pM to about 200 pM, about 30 pM to about 150 pM, about 30 pM to about100 pM, about 30 pM to about 90 pM, about 30 pM to about 80 pM, about 30pM to about 70 pM, about 30 pM to about 60 pM, about 30 pM to about 50pM, about 30 pM to about 40 pM, about 40 pM to about 30 nM, about 40 pMto about 25 nM, about 40 pM to about 30 nM, about 40 pM to about 15 nM,about 40 pM to about 10 nM, about 40 pM to about 5 nM, about 40 pM toabout 2 nM, about 40 pM to about 1 nM, about 40 pM to about 950 pM,about 40 pM to about 900 pM, about 40 pM to about 850 pM, about 40 pM toabout 800 pM, about 40 pM to about 750 pM, about 40 pM to about 700 pM,about 40 pM to about 650 pM, about 40 pM to about 600 pM, about 40 pM toabout 550 pM, about 40 pM to about 500 pM, about 40 pM to about 450 pM,about 40 pM to about 400 pM, about 40 pM to about 350 pM, about 40 pM toabout 300 pM, about 40 pM to about 250 pM, about 40 pM to about 200 pM,about 40 pM to about 150 pM, about 40 pM to about 100 pM, about 40 pM toabout 90 pM, about 40 pM to about 80 pM, about 40 pM to about 70 pM,about 40 pM to about 60 pM, about 40 pM to about 50 pM, about 50 pM toabout 30 nM, about 50 pM to about 25 nM, about 50 pM to about 30 nM,about 50 pM to about 15 nM, about 50 pM to about 10 nM, about 50 pM toabout 5 nM, about 50 pM to about 2 nM, about 50 pM to about 1 nM, about50 pM to about 950 pM, about 50 pM to about 900 pM, about 50 pM to about850 pM, about 50 pM to about 800 pM, about 50 pM to about 750 pM, about50 pM to about 700 pM, about 50 pM to about 650 pM, about 50 pM to about600 pM, about 50 pM to about 550 pM, about 50 pM to about 500 pM, about50 pM to about 450 pM, about 50 pM to about 400 pM, about 50 pM to about350 pM, about 50 pM to about 300 pM, about 50 pM to about 250 pM, about50 pM to about 200 pM, about 50 pM to about 150 pM, about 50 pM to about100 pM, about 50 pM to about 90 pM, about 50 pM to about 80 pM, about 50pM to about 70 pM, about 50 pM to about 60 pM, about 60 pM to about 30nM, about 60 pM to about 25 nM, about 60 pM to about 30 nM, about 60 pMto about 15 nM, about 60 pM to about 10 nM, about 60 pM to about 5 nM,about 60 pM to about 2 nM, about 60 pM to about 1 nM, about 60 pM toabout 950 pM, about 60 pM to about 900 pM, about 60 pM to about 850 pM,about 60 pM to about 800 pM, about 60 pM to about 750 pM, about 60 pM toabout 700 pM, about 60 pM to about 650 pM, about 60 pM to about 600 pM,about 60 pM to about 550 pM, about 60 pM to about 500 pM, about 60 pM toabout 450 pM, about 60 pM to about 400 pM, about 60 pM to about 350 pM,about 60 pM to about 300 pM, about 60 pM to about 250 pM, about 60 pM toabout 200 pM, about 60 pM to about 150 pM, about 60 pM to about 100 pM,about 60 pM to about 90 pM, about 60 pM to about 80 pM, about 60 pM toabout 70 pM, about 70 pM to about 30 nM, about 70 pM to about 25 nM,about 70 pM to about 30 nM, about 70 pM to about 15 nM, about 70 pM toabout 10 nM, about 70 pM to about 5 nM, about 70 pM to about 2 nM, about70 pM to about 1 nM, about 70 pM to about 950 pM, about 70 pM to about900 pM, about 70 pM to about 850 pM, about 70 pM to about 800 pM, about70 pM to about 750 pM, about 70 pM to about 700 pM, about 70 pM to about650 pM, about 70 pM to about 600 pM, about 70 pM to about 550 pM, about70 pM to about 500 pM, about 70 pM to about 450 pM, about 70 pM to about400 pM, about 70 pM to about 350 pM, about 70 pM to about 300 pM, about70 pM to about 250 pM, about 70 pM to about 200 pM, about 70 pM to about150 pM, about 70 pM to about 100 pM, about 70 pM to about 90 pM, about70 pM to about 80 pM, about 80 pM to about 30 nM, about 80 pM to about25 nM, about 80 pM to about 30 nM, about 80 pM to about 15 nM, about 80pM to about 10 nM, about 80 pM to about 5 nM, about 80 pM to about 2 nM,about 80 pM to about 1 nM, about 80 pM to about 950 pM, about 80 pM toabout 900 pM, about 80 pM to about 850 pM, about 80 pM to about 800 pM,about 80 pM to about 750 pM, about 80 pM to about 700 pM, about 80 pM toabout 650 pM, about 80 pM to about 600 pM, about 80 pM to about 550 pM,about 80 pM to about 500 pM, about 80 pM to about 450 pM, about 80 pM toabout 400 pM, about 80 pM to about 350 pM, about 80 pM to about 300 pM,about 80 pM to about 250 pM, about 80 pM to about 200 pM, about 80 pM toabout 150 pM, about 80 pM to about 100 pM, about 80 pM to about 90 pM,about 90 pM to about 30 nM, about 90 pM to about 25 nM, about 90 pM toabout 30 nM, about 90 pM to about 15 nM, about 90 pM to about 10 nM,about 90 pM to about 5 nM, about 90 pM to about 2 nM, about 90 pM toabout 1 nM, about 90 pM to about 950 pM, about 90 pM to about 900 pM,about 90 pM to about 850 pM, about 90 pM to about 800 pM, about 90 pM toabout 750 pM, about 90 pM to about 700 pM, about 90 pM to about 650 pM,about 90 pM to about 600 pM, about 90 pM to about 550 pM, about 90 pM toabout 500 pM, about 90 pM to about 450 pM, about 90 pM to about 400 pM,about 90 pM to about 350 pM, about 90 pM to about 300 pM, about 90 pM toabout 250 pM, about 90 pM to about 200 pM, about 90 pM to about 150 pM,about 90 pM to about 100 pM, about 100 pM to about 30 nM, about 100 pMto about 25 nM, about 100 pM to about 30 nM, about 100 pM to about 15nM, about 100 pM to about 10 nM, about 100 pM to about 5 nM, about 100pM to about 2 nM, about 100 pM to about 1 nM, about 100 pM to about 950pM, about 100 pM to about 900 pM, about 100 pM to about 850 pM, about100 pM to about 800 pM, about 100 pM to about 750 pM, about 100 pM toabout 700 pM, about 100 pM to about 650 pM, about 100 pM to about 600pM, about 100 pM to about 550 pM, about 100 pM to about 500 pM, about100 pM to about 450 pM, about 100 pM to about 400 pM, about 100 pM toabout 350 pM, about 100 pM to about 300 pM, about 100 pM to about 250pM, about 100 pM to about 200 pM, about 100 pM to about 150 pM, about150 pM to about 30 nM, about 150 pM to about 25 nM, about 150 pM toabout 30 nM, about 150 pM to about 15 nM, about 150 pM to about 10 nM,about 150 pM to about 5 nM, about 150 pM to about 2 nM, about 150 pM toabout 1 nM, about 150 pM to about 950 pM, about 150 pM to about 900 pM,about 150 pM to about 850 pM, about 150 pM to about 800 pM, about 150 pMto about 750 pM, about 150 pM to about 700 pM, about 150 pM to about 650pM, about 150 pM to about 600 pM, about 150 pM to about 550 pM, about150 pM to about 500 pM, about 150 pM to about 450 pM, about 150 pM toabout 400 pM, about 150 pM to about 350 pM, about 150 pM to about 300pM, about 150 pM to about 250 pM, about 150 pM to about 200 pM, about200 pM to about 30 nM, about 200 pM to about 25 nM, about 200 pM toabout 30 nM, about 200 pM to about 15 nM, about 200 pM to about 10 nM,about 200 pM to about 5 nM, about 200 pM to about 2 nM, about 200 pM toabout 1 nM, about 200 pM to about 950 pM, about 200 pM to about 900 pM,about 200 pM to about 850 pM, about 200 pM to about 800 pM, about 200 pMto about 750 pM, about 200 pM to about 700 pM, about 200 pM to about 650pM, about 200 pM to about 600 pM, about 200 pM to about 550 pM, about200 pM to about 500 pM, about 200 pM to about 450 pM, about 200 pM toabout 400 pM, about 200 pM to about 350 pM, about 200 pM to about 300pM, about 200 pM to about 250 pM, about 300 pM to about 30 nM, about 300pM to about 25 nM, about 300 pM to about 30 nM, about 300 pM to about 15nM, about 300 pM to about 10 nM, about 300 pM to about 5 nM, about 300pM to about 2 nM, about 300 pM to about 1 nM, about 300 pM to about 950pM, about 300 pM to about 900 pM, about 300 pM to about 850 pM, about300 pM to about 800 pM, about 300 pM to about 750 pM, about 300 pM toabout 700 pM, about 300 pM to about 650 pM, about 300 pM to about 600pM, about 300 pM to about 550 pM, about 300 pM to about 500 pM, about300 pM to about 450 pM, about 300 pM to about 400 pM, about 300 pM toabout 350 pM, about 400 pM to about 30 nM, about 400 pM to about 25 nM,about 400 pM to about 30 nM, about 400 pM to about 15 nM, about 400 pMto about 10 nM, about 400 pM to about 5 nM, about 400 pM to about 2 nM,about 400 pM to about 1 nM, about 400 pM to about 950 pM, about 400 pMto about 900 pM, about 400 pM to about 850 pM, about 400 pM to about 800pM, about 400 pM to about 750 pM, about 400 pM to about 700 pM, about400 pM to about 650 pM, about 400 pM to about 600 pM, about 400 pM toabout 550 pM, about 400 pM to about 500 pM, about 500 pM to about 30 nM,about 500 pM to about 25 nM, about 500 pM to about 30 nM, about 500 pMto about 15 nM, about 500 pM to about 10 nM, about 500 pM to about 5 nM,about 500 pM to about 2 nM, about 500 pM to about 1 nM, about 500 pM toabout 950 pM, about 500 pM to about 900 pM, about 500 pM to about 850pM, about 500 pM to about 800 pM, about 500 pM to about 750 pM, about500 pM to about 700 pM, about 500 pM to about 650 pM, about 500 pM toabout 600 pM, about 500 pM to about 550 pM, about 600 pM to about 30 nM,about 600 pM to about 25 nM, about 600 pM to about 30 nM, about 600 pMto about 15 nM, about 600 pM to about 10 nM, about 600 pM to about 5 nM,about 600 pM to about 2 nM, about 600 pM to about 1 nM, about 600 pM toabout 950 pM, about 600 pM to about 900 pM, about 600 pM to about 850pM, about 600 pM to about 800 pM, about 600 pM to about 750 pM, about600 pM to about 700 pM, about 600 pM to about 650 pM, about 700 pM toabout 30 nM, about 700 pM to about 25 nM, about 700 pM to about 30 nM,about 700 pM to about 15 nM, about 700 pM to about 10 nM, about 700 pMto about 5 nM, about 700 pM to about 2 nM, about 700 pM to about 1 nM,about 700 pM to about 950 pM, about 700 pM to about 900 pM, about 700 pMto about 850 pM, about 700 pM to about 800 pM, about 700 pM to about 750pM, about 800 pM to about 30 nM, about 800 pM to about 25 nM, about 800pM to about 30 nM, about 800 pM to about 15 nM, about 800 pM to about 10nM, about 800 pM to about 5 nM, about 800 pM to about 2 nM, about 800 pMto about 1 nM, about 800 pM to about 950 pM, about 800 pM to about 900pM, about 800 pM to about 850 pM, about 900 pM to about 30 nM, about 900pM to about 25 nM, about 900 pM to about 30 nM, about 900 pM to about 15nM, about 900 pM to about 10 nM, about 900 pM to about 5 nM, about 900pM to about 2 nM, about 900 pM to about 1 nM, about 900 pM to about 950pM, about 1 nM to about 30 nM, about 1 nM to about 25 nM, about 1 nM toabout 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about1 nM to about 5 nM, about 2 nM to about 30 nM, about 2 nM to about 25nM, about 2 nM to about 20 nM, about 2 nM to about 15 nM, about 2 nM toabout 10 nM, about 2 nM to about 5 nM, about 4 nM to about 30 nM, about4 nM to about 25 nM, about 4 nM to about 20 nM, about 4 nM to about 15nM, about 4 nM to about 10 nM, about 4 nM to about 5 nM, about 5 nM toabout 30 nM, about 5 nM to about 25 nM, about 5 nM to about 20 nM, about5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 30nM, about 10 nM to about 25 nM, about 10 nM to about 20 nM, about 10 nMto about 15 nM, about 15 nM to about 30 nM, about 15 nM to about 25 nM,about 15 nM to about 20 nM, about 20 nM to about 30 nM, and about 20 nMto about 25 nM).

Any of the target-binding domains described herein can bind to itstarget with a K_(D) of between about 1 nM to about 10 nM (e.g., about 1nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM,about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM toabout 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM,about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM toabout 7 nM, about 3 nM to about 6 nM, about 3 nM to about 5 nM, about 3nM to about 4 nM, about 4 nM to about 10 nM, about 4 nM to about 9 nM,about 4 nM to about 8 nM, about 4 nM to about 7 nM, about 4 nM to about6 nM, about 4 nM to about 5 nM, about 5 nM to about 10 nM, about 5 nM toabout 9 nM, about 5 nM to about 8 nM, about 5 nM to about 7 nM, about 5nM to about 6 nM, about 6 nM to about 10 nM, about 6 nM to about 9 nM,about 6 nM to about 8 nM, about 6 nM to about 7 nM, about 7 nM to about10 nM, about 7 nM to about 9 nM, about 7 nM to about 8 nM, about 8 nM toabout 10 nM, about 8 nM to about 9 nM, and about 9 nM to about 10 nM).

A variety of different methods known in the art can be used to determinethe K_(D) values of any of the antigen-binding protein constructsdescribed herein (e.g., an electrophoretic mobility shift assay, afilter binding assay, surface plasmon resonance, and a biomolecularbinding kinetics assay, etc.).

Antigen-Binding Domains

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtarget-binding domain bind specifically to the same antigen. In someembodiments of these single-chain chimeric polypeptides, the firsttarget-binding domain and the second target-binding domain bindspecifically to the same epitope. In some embodiments of thesesingle-chain chimeric polypeptides, the first target-binding domain andthe second target-binding domain include the same amino acid sequence.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtarget-binding domain bind specifically to different antigens.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, one or both of the first target-binding domain and thesecond target-binding domain is an antigen-binding domain. In someembodiments of any of the single-chain chimeric polypeptides describedherein, the first target-binding domain and the second target-bindingdomain are each an antigen-binding domain.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the antigen-binding domain includes or is a scFv or asingle domain antibody (e.g., a V_(H)H or a V_(NAR) domain).

In some examples, an antigen-binding domain (e.g., any of theantigen-binding domains described herein) can bind specifically to anyone of CD16a (see, e.g., those described in U.S. Pat. No. 9,035,026),CD28 (see, e.g., those described in U.S. Pat. No. 7,723,482), CD3 (see,e.g., those described in U.S. Pat. No. 9,226,962), CD33 (see, e.g.,those described in U.S. Pat. No. 8,759,494), CD20 (see, e.g., thosedescribed in WO 2014/026054), CD19 (see, e.g., those described in U.S.Pat. No. 9,701,758), CD22 (see, e.g., those described in WO2003/104425), CD123 (see, e.g., those described in WO 2014/130635),IL-1R (see, e.g., those described in U.S. Pat. No. 8,741,604), IL-1(see, e.g., those described in WO 2014/095808), VEGF (see, e.g., thosedescribed in U.S. Pat. No. 9,090,684), IL-6R (see, e.g., those describedin U.S. Pat. No. 7,482,436), IL-4 (see, e.g., those described in U.S.Patent Application Publication No. 2012/0171197), IL-10 (see, e.g.,those described in U.S. Patent Application Publication No.2016/0340413), PDL-1 (see, e.g., those described in Drees et al.,Protein Express. Purif. 94:60-66, 2014), TIGIT (see, e.g., thosedescribed in U.S. Patent Application Publication No. 2017/0198042), PD-1(see, e.g., those described in U.S. Pat. No. 7,488,802), TIM3 (see,e.g., those described in U.S. Pat. No. 8,552,156), CTLA4 (see, e.g.,those described in WO 2012/120125), MICA (see, e.g., those described inWO 2016/154585), MICB (see, e.g., those described in U.S. Pat. No.8,753,640), IL-6 (see, e.g., those described in Gejima et al., HumanAntibodies 11(4):121-129, 2002), IL-8 (see, e.g., those described inU.S. Pat. No. 6,117,980), TNFα (see, e.g., those described in Geng etal., Immunol. Res. 62(3):377-385, 2015), CD26a (see, e.g., thosedescribed in WO 2017/189526), CD36 (see, e.g., those described in U.S.Patent Application Publication No. 2015/0259429), ULBP2 (see, e.g.,those described in U.S. Pat. No. 9,273,136), CD30 (see, e.g., thosedescribed in Homach et al., Scand. J. Immunol. 48(5):497-501, 1998),CD200 (see, e.g., those described in U.S. Pat. No. 9,085,623), IGF-1R(see, e.g., those described in U.S. Patent Application Publication No.2017/0051063), MUC4AC (see, e.g., those described in WO 2012/170470),MUC5AC (see, e.g., those described in U.S. Pat. No. 9,238,084), Trop-2(see, e.g., those described in WO 2013/068946), CMET (see, e.g., thosedescribed in Edwardraja et al., Biotechnol. Bioeng. 106(3):367-375,2010), EGFR (see, e.g., those described in Akbari et al., Protein Expr.Purif. 127:8-15, 2016), HER1 (see, e.g., those described in U.S. PatentApplication Publication No. 2013/0274446), HER2 (see, e.g., thosedescribed in Cao et al., Biotechnol. Lett. 37(7):1347-1354, 2015), HER3(see, e.g., those described in U.S. Pat. No. 9,505,843), PSMA (see,e.g., those described in Parker et al., Protein Expr. Purif.89(2):136-145, 2013), CEA (see, e.g., those described in WO1995/015341), B7H3 (see, e.g., those described in U.S. Pat. No.9,371,395), EPCAM (see, e.g., those described in WO 2014/159531), BCMA(see, e.g., those described in Smith et al., Mol. Ther. 26(6):1447-1456,2018), P-cadherin (see, e.g., those described in U.S. Pat. No.7,452,537), CEACAM5 (see, e.g., those described in U.S. Pat. No.9,617,345), a UL16-binding protein (see, e.g., those described in WO2017/083612), HLA-DR (see, e.g., Pistillo et al., Exp. Clin.Immunogenet. 14(2):123-130, 1997), DLL4 (see, e.g., those described inWO 2014/007513), TYRO3 (see, e.g., those described in WO 2016/166348),AXL (see, e.g., those described in WO 2012/175692), MER (see, e.g.,those described in WO 2016/106221), CD122 (see, e.g., those described inU.S. Patent Application Publication No. 2016/0367664), CD155 (see, e.g.,those described in WO 2017/149538), or PDGFDD (see, e.g., thosedescribed in U.S. Pat. No. 9,441,034).

The antigen-binding domains present in any of the single-chain chimericpolypeptides described herein are each independently selected from thegroup consisting of: a VHH domain, a VNAR domain, and a scFv.

In some embodiments, the first target binding domain, the secondtarget-binding domain, and/or one or more of the one or more additionalantigen-binding domains can be an anti-CD3 scFv. In some embodiments,the anti-CD3 scFv can include a heavy chain variable domain including asequence that is at least 70% identical (e.g., at least 75% identical,at least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, at least 99% identical, or 100% identical) toQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCL DYWGQGTTLTVSS (SEQID NO: 16) and/or a light chain variable domain including a sequencethat is at least 70% identical (e.g., at least 75% identical, at least80% identical, at least 85% identical, at least 90% identical, at least95% identical, at least 99% identical, or 100% identical) toQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 17).In some embodiments, a scFv (e.g., any of the scFvs described herein)can include a linker sequence (e.g., any of the exemplary linkersequences described herein or known in the art) between the heavy chainvariable domain and the light chain variable domain. In someembodiments, the anti-CD3 scFv can include a heavy chain variable domainencoded by a nucleic acid including a sequence that is at least 70%identical (e.g., at least 75% identical, at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical, atleast 99% identical, or 100% identical) toCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC (SEQ ID NO: 18), and/or alight chain variable domain encoded by a nucleic acid including asequence that is at least 70% identical (e.g., at least 75% identical,at least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, at least 99% identical, or 100% identical) to

(SEQ ID NO: 19) CAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGT.

In some embodiments, an anti-CD3 scFv can include a sequence that is atleast 70% identical (e.g., at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 99% identical, or 100% identical) toQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (SEQ ID NO: 20). In some embodiments, ananti-CD3 scFv can include the six CDRs present in SEQ ID NO: 20.

In some embodiments, an anti-CD3 scFv can include a sequence encoded bya nucleic acid sequence that is at least 70% identical (e.g., at least75% identical, at least 80% identical, at least 85% identical, at least90% identical, at least 95% identical, at least 99% identical, or 100%identical) to

(SEQ ID NO: 21) CAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGTGGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGGAGGTAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC.

In some embodiments, the first target binding domain, the secondtarget-binding domain, and/or one or more of the one or more additionalantigen-binding domains can be an anti-CD28 scFv. In some embodiments,the anti-CD28 scFv can include a heavy chain variable domain including asequence that is at least 70% identical (e.g., at least 75% identical,at least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, at least 99% identical, or 100% identical) toDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR (SEQ ID NO: 22)and/or a light chain variable domain including a sequence that is atleast 70% identical (e.g., at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 99% identical, or 100% identical) toVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRG TTLTVSS (SEQ IDNO: 23). In some embodiments, the anti-CD28 scFv can include a heavychain variable domain encoded by a nucleic acid including a sequencethat is at least 70% identical (e.g., at least 75% identical, at least80% identical, at least 85% identical, at least 90% identical, at least95% identical, at least 99% identical, or 100% identical) toGACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAGACC AAGCGG (SEQ ID NO:24), and/or a light chain variable domain encoded by a nucleic acidincluding a sequence that is at least 70% identical (e.g., at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, at least 95% identical, at least 99% identical, or 100%identical) to

(SEQ ID NO: 25) GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGCCGGGGAACTACTTTAACAGTGAGCTCC.

In some embodiments, an anti-CD28 scFv can include a sequence that is atleast 70% identical (e.g., at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 99% identical, or 100% identical) toVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR (SEQ ID NO: 26). In some embodiments, an anti-CD28scFv can include the six CDRs present in SEQ ID NO: 26.

In some embodiments, an anti-CD28 scFv can include a sequence encoded bya nucleic acid sequence that is at least 70% identical (e.g., at least75% identical, at least 80% identical, at least 85% identical, at least90% identical, at least 95% identical, at least 99% identical, or 100%identical) to

(SEQ ID NO: 27) GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGCCGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGCGACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAGACCAAGCGG.

In some embodiments, any of the antigen-binding domains described hereinis a BiTe, a (scFv)₂, a nanobody, a nanobody-HSA, a DART, a TandAb, ascDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HAS,or a tandem-scFv. Additional examples of antigen-binding domains thatcan be used in any of the single-chain chimeric polypeptide are known inthe art.

A VHH domain is a single monomeric variable antibody domain that can befound in camelids. A V_(NAR) domain is a single monomeric variableantibody domain that can be found in cartilaginous fish. Non-limitingaspects of VHH domains and V_(NAR) domains are described in, e.g.,Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst etal., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., TrendsBiotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174,2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014;Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delicet al., Trends Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J.Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends Biochem. Sci.26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013;Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove etal., EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin.Investig. Drugs 10:1212-1224, 2009; Vincke et al., Methods Mol. Biol.911:15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol.198:157-174, 2009.

In some embodiments, each of the antigen-binding domains in thesingle-chain chimeric polypeptides described herein are both VHHdomains, or at least one antigen-binding domain is a VHH domain. In someembodiments, each of the antigen-binding domains in the single-chainchimeric polypeptides described herein are both VNAR domains, or atleast one antigen-binding domain is a VNAR domain. In some embodiments,each of the antigen-binding domains in the single-chain chimericpolypeptides described herein are both scFv domains, or at least oneantigen-binding domain is a scFv domain. DARTs are described in, e.g.,Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

In some embodiments of any of the antigen-binding domains describedherein can bind to an antigen selected from the group consisting of: aprotein, a carbohydrate, a lipid, and a combination thereof.

Additional examples and aspects of antigen-binding domains are known inthe art.

Soluble Interleukin or Cytokine Protein

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, one or both of the first target-binding domain and thesecond target-binding domain can be a soluble interleukin protein,soluble cytokine protein, or soluble cell surface protein. In someembodiments, the soluble interleukin or soluble cytokine protein, isselected from the group of: IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L. Non-limiting examples ofsoluble IL-2, IL-3, IL-7, IL-8, IL-10, IL-15, IL-17, IL-18, IL-21,PDGF-DD, SCF, and FLT3L are provided below.

Human Soluble IL-2 (SEQ ID NO: 28)aptssstkkt qlqlehllld lqmilnginn yknpkltrml tfkfympkkatelkhlqcle eelkpleevl nlaqsknfhl rprdlisnin vivlelkgsettfmceyade tativeflnr witfcqsiis tlt Human Soluble IL-3 (SEQ ID NO: 29)apmtqttplkt swvncsnmid eiithlkqpp lplldfnnln gedqdilmennlrrpnleaf nravkslqna saiesilknl lpclplataa ptrhpihikdgdwnefrrkl tfylktlena qaqqttlsla if Human Soluble IL-7 (SEQ ID NO: 30)dcdiegkdgkqyesv lmvsidqlld smkeigsncl nnefnffkrh icdankegmflfraarklrq flkmnstgdf dlhllkvseg ttillnctgq vkgrkpaalgeaqptkslee nkslkeqkkl ndlcflkrll qeiktcwnki lmgtkeh Human Soluble IL-8(SEQ ID NO: 31) egavlprsak elrcqcikty skpfhpkfik elrviesgph canteiivklsdgrelcldp kenwvqrvve kflkraens Human Soluble IL-10 (SEQ ID NO: 32)spgqgtqsensc thfpgnlpnm lrdlrdafsr vktffqmkdq ldnlllkeslledfkgylgc qalsemiqfy leevmpqaen qdpdikahvn slgenlktlrlrlrrchrfl pcenkskave qvknafnklq ekgiykamse fdifinyiea ymtmkirnHuman Soluble IL-12β (p40) (SEQ ID NO: 33)IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSNucleic Acid Encoding Human Soluble IL-12β (p40) (SEQ ID NO: 34)ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC Human Soluble IL-12α (p35) (SEQ ID NO: 35)RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASNucleic Acid Encoding Human Soluble IL-12α (p35) (SEQ ID NO: 36)CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAAC GCCAGCExemplary Human Soluble IL-12 (SEQ ID NO: 37)IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASNucleic Acid Encoding Exemplary Human Soluble IL-12 (SEQ ID NO: 38)ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC Human Soluble IL-15(SEQ ID NO: 39) Nwvnvisdlkki edliqsmhid atlytesdvh psckvtamkc fllelqvislesgdasihdt venliilann slssngnvte sgckeceele eknikeflqs fvhivqmfin tsHuman Soluble IL-17 (SEQ ID NO: 40)gitiprn pgcpnsedkn fprtvmvnln ihnrntntnp krssdyynrstspwnlhrne dperypsviw eakcrhlgci nadgnvdyhm nsvpiqqeilvlrrepphcp nsfrlekilv svgctcvtpi vhhva Human Soluble IL-18(SEQ ID NO: 41)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDNucleic Acid Encoding Human Soluble IL-18 (SEQ ID NO: 42)TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT Human Soluble IL-21(SEQ ID NO: 43) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS Nucleic Acid Encoding Human Soluble IL-21(SEQ ID NO: 44) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC Human Soluble PDGF-DD(SEQ ID NO: 45) rdtsatpqsasi kalrnanlrr desnhltdly rrdetiqvkg ngyvqsprfpnsyprnlllt wrlhsqentr iqlvfdnqfg leeaendicr ydfvevedisetstiirgrw cghkevppri ksrtnqikit fksddyfvak pgfkiyyslledfqpaaase tnwesvtssi sgvsynspsv tdptliadal dkkiaefdtvedllkyfnpe swqedlenmy ldtpryrgrs yhdrkskvdl drlnddakrysctprnysvn ireelklanv vffprcllvq rcggncgcgt vnwrsctcnsgktvkkyhev lqfepghikr rgraktmalv diqldhherc dcicssrppr Human Soluble SCF(SEQ ID NO: 46)egicrnrvtnnvkdv tklvanlpkd ymitlkyvpg mdvlpshcwi semvvqlsdsltdlldkfsn iseglsnysi idklvnivdd lvecvkenss kdlkksfkspeprlftpeef frifnrsida fkdfvvaset sdcvvsstls pekdsrvsvtkpfmlppvaa sslrndssss nrkaknppgd sslhwaamal palfsliigfafgalywkkr qpsltraven iqineednei smlqekeref qev Human Soluble FLT3L(SEQ ID NO: 47)tqdcsfqhspissd favkirelsd yllqdypvtv asnlqdeelc gglwrlvlaqrwmerlktva gskmqgller vnteihfvtk cafqpppscl rfvqtnisrllqetseqlva lkpwitrqnf srclelqcqp dsstlpppws prpleataptapqpplllll llpvglllla aawclhwqrt rrrtprpgeq vppvpspqdl llveh

Exemplary soluble cell surface proteins include soluble MICA, MICB, anda ULP16 binding protein (e.g., ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, orULBP6). Exemplary sequences for soluble MICA, MICB, ULBP1, ULBP2, ULBP3,ULBP4, ULBP5, and ULBP6 are listed below.

Human Soluble MICA (SEQ ID NO: 48)ephslry nltvlswdgs vqsgfltevh idgqpflrcdrqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtlahikdqkegl hslqeirvce ihednstrss qhfyydgelflsqnletkew tmpqssraqt lamnvrnflk edamktkthyhamhadclqe lrrylksgvv lrrtvppmvn vtrseasegnitvtcrasgf ypwnitlswr qdgvslshdt qqwgdvlpdgngtyqtwvat ricqgeeqrf tcymehsgnh sthpvpsgkvlvlqshwqtf hvsavaaaai fviiifyvrc ckkktsaaegpelvslqvld qhpvgtsdhr datqlgfqpl msdlgstgst ega Human Soluble MICB(SEQ ID NO: 49) aephslry nlmvlsqdes vqsgflaegh ldgqpflrydrqkrrakpqg qwaedvlgak twdtetedlt engqdlrrtlthikdqkggl hslqeirvce ihedsstrgs rhfyydgelflsqnletqes tvpqssraqt lamnvtnfwk edamktkthyramqadclqk lqrylksgva irrtvppmvn vtcsevsegnitvtcrassf yprnitltwr qdgvslshnt qqwgdvlpdgngtyqtwvat rirqgeeqrf tcymehsgnh gthpvpsgkvlvlqsqrtdf pyvsaampcf viiiilcvpc ckkktsaaegpelvslqvld qhpvgtgdhr daaqlgfqpl msatgstgst ega Human Soluble ULBP1(SEQ ID NO: 50) wvdthclcydfiit pksrpepqwc evqglvderp flhydcvnhkakafaslgkk vnvtktweeq tetlrdvvdf lkgqlldiqvenlipieplt lqarmscehe ahghgrgswq flfngqkfllfdsnnrkwta lhpgakkmte kweknrdvtm ffqkislgdc kmwleeflmy weqmldptkp pslapgHuman Soluble ULBP2 (SEQ ID NO: 51)gradphslcyditvi pkfrpgprwc avqgqvdekt flhydcgnktvtpvsplgkk lnvttawkaq npvlrevvdi lteqlrdiqlenytpkeplt lqarmsceqk aeghssgswq fsfdgqifllfdsekrmwtt vhpgarkmke kwendkvvam sfhyfsmgdc igwledflmg mdstlepsag aplamsHuman Soluble ULBP3 (SEQ ID NO: 52)dahslwynfti ihlprhgqqw cevqsqvdqk nflsydcgsdkvlsmghlee qlyatdawgk qlemlrevgq rlrleladteledftpsgpl tlqvrmscec eadgyirgsw qfsfdgrkfllfdsnnrkwt vvhagarrmk ekwekdsglt tffkmvsmrdckswlrdflm hrkkrlepta pptmapg Human Soluble ULBP4 (SEQ ID NO: 53)hslcfnftik slsrpgqpwc eaqvflnknl flqynsdnnmvkplgllgkk vyatstwgel tqtlgevgrd lrmllcdikpqiktsdpstl qvemfcqrea erctgaswqf atngeksllfdamnmtwtvi nheaskiket wkkdrgleky frklskgdcdhwlreflghw eampeptvsp vnasdihwss sslpdrwiilgafillvlmg ivlicvwwqn gewqaglwpl rts Human Soluble ULBP5 (SEQ ID NO: 54)gladp hslcyditvi pkfrpgprwc avqgqvdekt flhydcgsktvtpvsplgkk lnvttawkaq npvlrevvdi lteqlldiqlenyipkeplt lqarmsceqk aeghgsgswq lsfdgqifllfdsenrmwtt vhpgarkmke kwendkdmtm sfhyismgdctgwledflmg mdstlepsag apptmssg Human Soluble ULBP6 (SEQ ID NO: 55)rrddp hslcyditvi pkfrpgprwc avqgqvdekt flhydcgnktvtpvsplgkk lnvtmawkaq npvlrevvdi lteqlldiqlenytpkeplt lqarmsceqk aeghssgswq fsidgqtfllfdsekrmwtt vhpgarkmke kwendkdvam sfhyismgdcigwledflmg mdstlepsag aplamssg

In some embodiments, a soluble IL-12 protein can include a firstsequence that is at least 70% identical (e.g., at least 75% identical,at least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, at least 99% identical, or 100% identical) toSEQ ID NO: 33, and a second sequence that is at least 70% identical(e.g., at least 75% identical, at least 80% identical, at least 85%identical, at least 90% identical, at least 95% identical, at least 99%identical, or 100% identical) to SEQ ID NO: 35. In some embodiments, thesoluble IL-12 can further include a linker sequence (e.g., any of theexemplary linker sequences described herein or known in the art) betweenthe first sequence and the second sequence.

In some embodiments, a soluble IL-12 protein is encoded by a firstnucleic acid encoding a first sequence at least 70% identical (e.g., atleast 75% identical, at least 80% identical, at least 85% identical, atleast 90% identical, at least 95% identical, at least 99% identical, or100% identical) to SEQ ID NO: 34, and a second nucleic acid sequenceencoding a second sequence that is at least 70% identical (e.g., atleast 75% identical, at least 80% identical, at least 85% identical, atleast 90% identical, at least 95% identical, at least 99% identical, or100% identical) to SEQ ID NO: 36. In some embodiments, the nucleic acidencoding a soluble IL-12 protein further includes a nucleic acidsequence encoding a linker sequence (e.g., any of the exemplary linkersequences described herein or known in the art) between the firstnucleic acid and the second nucleic acid.

In some embodiments, a soluble IL-12 protein includes a sequence that isat least 70% identical (e.g., at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 99% identical, or 100% identical) to SEQ ID NO: 37.In some embodiments, a soluble IL-12 protein is encoded by a nucleicacid including a sequence at least 70% identical (e.g., at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, at least 95% identical, at least 99% identical, or 100%identical) to SEQ ID NO: 38.

In some embodiments, a soluble IL-18 protein can include a sequence thatis at least 70% identical (e.g., at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 99% identical, or 100% identical) to SEQ ID NO: 41.In some embodiments, a soluble IL-18 protein is encoded by a nucleicacid including a sequence at least 70% identical (e.g., at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, at least 95% identical, at least 99% identical, or 100%identical) to SEQ ID NO: 42.

In some embodiments, a soluble IL-21 protein can include a sequence thatis at least 70% identical (e.g., at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 99% identical, or 100% identical) to SEQ ID NO: 43.In some embodiments, a soluble IL-21 protein is encoded by a nucleicacid including a sequence at least 70% identical (e.g., at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, at least 95% identical, at least 99% identical, or 100%identical) to SEQ ID NO: 44.

Additional examples of soluble interleukin proteins and soluble cytokineproteins are known in the art.

Soluble Receptor

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, one or both of the first target-binding domain and thesecond target-binding domain is a soluble interleukin receptor, asoluble cytokine receptor, or a soluble cell surface receptor. In someembodiments, the soluble receptor is a soluble TGF-β receptor II(TGF-βRII) (see, e.g., those described in Yung et al., Am. J. Resp.Crit. Care Med. 194(9):1140-1151, 2016), a soluble TGF-βRIII (see, e.g.,those described in Heng et al., Placenta 57:320, 2017), or a solubleNKG2D (see, e.g., Cosman et al., Immunity 14(2):123-133, 2001; Costa etal., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi:10.3389/fimmu.2018.01150). In some embodiments, the soluble cell surfacereceptor is a soluble NKp30 (see, e.g., Costa et al., Front. Immunol.,Vol. 9, Article 1150, May 29, 2018; doi: 10.3389/fimmu.2018.01150), asoluble NKp44 (see, e.g., those described in Costa et al., Front.Immunol., Vol. 9, Article 1150, May 29, 2018; doi:10.3389/fimmu.2018.01150), a soluble NKp46 (see, e.g., Mandelboim etal., Nature 409:1055-1060, 2001; Costa et al., Front. Immunol., Vol. 9,Article 1150, May 29, 2018; doi: 10.3389/fimmu.2018.01150), a solubleDNAM1 (see, e.g., those described in Costa et al., Front. Immunol., Vol.9, Article 1150, May 29, 2018; doi: 10.3389/fimmu.2018.01150), a scMHCI(see, e.g., those described in Washburn et al., PLoS One 6(3):e18439,2011), a scMHCII (see, e.g., those described in Bishwajit et al.,Cellular Immunol. 170(1):25-33, 1996), a scTCR (see, e.g., thosedescribed in Weber et al., Nature 356(6372):793-796, 1992), a solubleCD155 (see, e.g., those described in Tahara-Hanaoka et al., Int.Immunol. 16(4):533-538, 2004), or a soluble CD28 (see, e.g., Hebbar etal., Clin. Exp. Immunol. 136:388-392, 2004).

In some embodiments, a soluble TGFβRII receptor can include a firstsequence that is at least 70% identical (e.g., at least 75% identical,at least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 56) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD,and a second sequence that is at least 70% identical (e.g., at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, at least 95% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 56) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD,In some embodiments, the soluble TGFβRII receptor can further include alinker sequence (e.g., any of the exemplary linker sequences describedherein or known in the art) between the first sequence and the secondsequence.

In some embodiments, a soluble TGFβRII receptor is encoded by a firstnucleic acid encoding a first sequence at least 70% identical (e.g., atleast 75% identical, at least 80% identical, at least 85% identical, atleast 90% identical, at least 95% identical, at least 99% identical, or100% identical) to:

(SEQ ID NO: 57) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT,and a second nucleic acid sequence encoding a second sequence that is atleast 70% identical (e.g., at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 57) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT.In some embodiments, the nucleic acid encoding a soluble TGFβRIIreceptor further includes a nucleic acid sequence encoding a linkersequence (e.g., any of the exemplary linker sequences described hereinor known in the art) between the first nucleic acid and the secondnucleic acid.

In some embodiments, a soluble TGFβRII receptor includes a sequence thatis at least 70% identical (e.g., at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 60) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.In some embodiments, a soluble TGFβRII receptor is encoded by a nucleicacid including a sequence at least 70% identical (e.g., at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, at least 95% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 61) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC.

Additional examples of soluble interleukin receptors and solublecytokine receptors are known in the art.

Additional Antigen-Binding Domains

Some embodiments of any of the single-chain chimeric polypeptidesdescribed herein can further include one or more (e.g., two, three,four, five, six, seven, eight, nine, or ten) additional target-bindingdomains (e.g., any of the exemplary target-binding domains describedherein or known in the art) at its N- and/or C-terminus.

In some embodiments, the single-chain chimeric polypeptides can includeone or more (e.g., two, three, four, five, six, seven, eight, nine, orten) additional target-binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) at itsN-terminus. In some embodiments, one of the one or more additionaltarget-binding domains (e.g., any of the exemplary target-bindingdomains described herein or known in the art) at the N-terminus of thesingle-chain chimeric polypeptide can directly abut the firsttarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art), the second target-binding domain(e.g., any of the exemplary target-binding domains described herein orknown in the art), or the soluble tissue factor domain (e.g., any of theexemplary soluble tissue factor domains described herein). In someembodiments, the single-chain chimeric polypeptide further includes alinker sequence (e.g., any of the exemplary linker sequences describedherein or known in the art) between one of the at least one additionaltarget-binding domains (e.g., any of the exemplary target-bindingdomains described herein or known in the art) at the N-terminus of thesingle-chain chimeric polypeptide and the first target-binding domain(e.g., any of the exemplary target-binding domains described herein orknown in the art), the second target-binding domain (e.g., any of theexemplary target-binding domains described herein or known in the art),or the soluble tissue factor domain (e.g., any of the exemplary solubletissue factor domains described herein).

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the single-chain chimeric polypeptide includes one ormore (e.g., two, three, four, five, six, seven, eight, nine, or ten)additional target-binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) at itsC-terminus. In some embodiments, one of the one or more additionaltarget-binding domains (e.g., any of the exemplary target-bindingdomains described herein or known in the art) at the C-terminus of thesingle-chain chimeric polypeptide directly abuts the firsttarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art), the second target-binding domain(e.g., any of the exemplary target-binding domains described herein orknown in the art), or the soluble tissue factor domain (e.g., any of theexemplary soluble tissue factor domains described herein or known in theart). In some embodiments, the single-chain chimeric polypeptide furthercomprises a linker sequence (e.g., any of the exemplary linker sequencesdescribed herein or known in the art) between one of the at least oneadditional target-binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) at theC-terminus of the single-chain chimeric polypeptide and the firsttarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art), the second target-binding domain(e.g., any of the exemplary target-binding domains described herein orknown in the art), or the soluble tissue factor domain (e.g., any of theexemplary soluble tissue factor domains described herein).

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the single-chain chimeric polypeptide comprises one ormore (e.g., two, three, four, five, six, seven, eight, nine, or ten)additional target binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) at itsN-terminus and its C-terminus. In some embodiments, one of the one ormore additional antigen binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) at theN-terminus of the single-chain chimeric polypeptide directly abuts thefirst target-binding domain (e.g., any of the exemplary target-bindingdomains described herein or known in the art), the second target-bindingdomain (e.g., any of the exemplary target-binding domains describedherein or known in the art), or the soluble tissue factor domain (e.g.,any of the exemplary soluble tissue factor domains described herein). Insome embodiments, the single-chain chimeric polypeptide further includesa linker sequence (e.g., any of the exemplary linker sequences describedherein or known in the art) between one of the one or more additionalantigen-binding domains (e.g., any of the exemplary target-bindingdomains described herein or known in the art) at the N-terminus and thefirst target-binding domain (e.g., any of the exemplary target-bindingdomains described herein or known in the art), the second target-bindingdomain (e.g., any of the exemplary target-binding domains describedherein or known in the art), or the soluble tissue factor domain (e.g.,any of the exemplary soluble tissue factor domains). In someembodiments, one of the one or more additional antigen binding domains(e.g., any of the exemplary target-binding domains described herein orknown in the art) at the C-terminus directly abuts the firsttarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art), the second target-binding domain(e.g., any of the exemplary target-binding domains described herein orknown in the art), or the soluble tissue factor domain (e.g., any of theexemplary soluble tissue factor domains). In some embodiments, thesingle-chain chimeric polypeptide further includes a linker sequence(e.g., any of the exemplary linker sequences described herein or knownin the art) between one of the one or more additional antigen-bindingdomains (e.g., any of the exemplary target-binding domains describedherein or known in the art) at the C-terminus and the firsttarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art), the second target-binding domain(e.g., any of the exemplary target-binding domains described herein orknown in the art), or the soluble tissue factor domain (e.g., any of theexemplary soluble tissue factor domains described herein).

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, two or more (e.g., three, four, five, six, seven,eight, nine, or ten) of the first target-binding domain (e.g., any ofthe exemplary target-binding domains described herein or known in theart), the second target-binding domain (e.g., any of the exemplarytarget-binding domains described herein or known in the art), and theone or more additional target-binding domains (e.g., any of theexemplary target-binding domains described herein or known in the art)bind specifically to the same antigen. In some embodiments, two or more(e.g., three, four, five, six, seven, eight, nine, or ten) of the firsttarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art), the second target-binding domain(e.g., any of the exemplary target-binding domains described herein orknown in the art), and the one or more additional target-binding domains(e.g., any of the exemplary target-binding domains described herein orknown in the art) bind specifically to the same epitope. In someembodiments, two or more (e.g., three, four, five, six, seven, eight,nine, or ten) of the first target-binding domain (e.g., any of theexemplary target-binding domains described herein or known in the art),the second target-binding domain (e.g., any of the exemplarytarget-binding domains described herein or known in the art), and theone or more additional target-binding domains (e.g., any of theexemplary target-binding domains described herein or known in the art)include the same amino acid sequence.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain (e.g., any of theexemplary target-binding domains described herein or known in the art),the second target-binding domain (e.g., any of the exemplarytarget-binding domains described herein or known in the art), and theone or more (e.g., two, three, four, five, six, seven, eight, nine, orten) additional target-binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) each bindspecifically to the same antigen. In some embodiments, the firsttarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art), the second target-binding domain(e.g., any of the exemplary target-binding domains described herein orknown in the art), and the one or more (e.g., two, three, four, five,six, seven, eight, nine, or ten) additional target-binding domains(e.g., any of the exemplary target-binding domains described herein orknown in the art) each bind specifically to the same epitope. In someembodiments, the first target-binding domain, the second target-bindingdomain, and the one or more (e.g., two, three, four, five, six, seven,eight, nine, or ten) additional target-binding domains each comprise thesame amino acid sequence.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain (e.g., any of theexemplary target-binding domains described herein or known in the art),the second target-binding domain (e.g., any of the exemplarytarget-binding domains described herein or known in the art), and theone or more (e.g., two, three, four, five, six, seven, eight, nine, orten) additional target-binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) bindspecifically to different antigens.

In some embodiments of any of the single-chain chimeric polypeptides,one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more target-binding domains is anantigen-binding domain (e.g., any of the exemplary antigen-bindingdomains described herein or known in the art). In some embodiments ofany of the single-chain chimeric polypeptides described herein, thefirst target-binding domain, the second target-binding domain, and theone or more additional target-binding domains are each anantigen-binding domain (e.g., any of the exemplary antigen-bindingdomains described herein or known in the art). In some embodiments, theantigen-binding domain can include a scFv or a single domain antibody.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, one or more (e.g., two, three, four, five, six, seven,eight, nine, or ten) of the first target-binding domain (e.g., any ofthe exemplary target-binding domains described herein or known in theart), the second target-binding domain (e.g., any of the exemplarytarget-binding domains described herein or known in the art), and theone or more target-binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) bindspecifically to a target selected from the group consisting of: CD16a,CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R,IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2,CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF-βRII), a ligand ofTGF-βRIII, a ligand of DNAM1, a ligand of NKp46, a ligand of NKp44, aligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand fora scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor forIL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, areceptor for IL-10, a receptor for IL-12, a receptor for IL-15, areceptor for IL-17, a receptor for IL-18, a receptor for IL-21, areceptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptorfor stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor forMICA, a receptor for MICB, a receptor for a ULP16-binding protein, areceptor for CD155, a receptor for CD122, and a receptor for CD28.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, one or more of the first target-binding domain (e.g.,any of the exemplary target-binding domains described herein or known inthe art), the second target-binding domain (e.g., any of the exemplarytarget-binding domains described herein or known in the art), and theone or more additional target-binding domains (e.g., any of theexemplary target-binding domains described herein or known in the art)is a soluble interleukin, a soluble cytokine protein, or a soluble cellsurface protein. Non-limiting examples of soluble interleukin proteinsand soluble cytokine proteins include: IL-1, IL-2, IL-3, IL-7, IL-8,IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.Non-limiting examples of soluble cell surface proteins include: MICA,MICB, and a ULP16-binding protein.

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, one or more of the first target-binding domain (e.g.,any of the exemplary target-binding domains described herein or known inthe art), the second target-binding domain (e.g., any of the exemplarytarget-binding domains described herein or known in the art), and theone or more additional target-binding domains (e.g., any of theexemplary target-binding domains described herein or known in the art)is a soluble interleukin receptor, a soluble cytokine receptor, or asoluble cell surface receptor. Non-limiting examples of solubleinterleukin receptors and soluble cytokine receptors include: a solubleTGF-β receptor II (TGF-βRII) and a soluble TGF-βRIII Non-limitingexamples of soluble cell surface receptors include: a soluble NKG2D, asoluble NK30, a soluble NKp44, a soluble NKp46, a soluble DNAM1, ascMHCI, a scMHCII, a scTCR, a soluble CD155, a soluble CD122, a solubleCD3, and a soluble CD28.

Signal Sequence

In some embodiments, a single-chain chimeric polypeptide includes asignal sequence at its N-terminal end. As will be understood by those ofordinary skill in the art, a signal sequence is an amino acid sequencethat is present at the N-terminus of a number of endogenously producedproteins that directs the protein to the secretory pathway (e.g., theprotein is directed to reside in certain intracellular organelles, toreside in the cell membrane, or to be secreted from the cell). Signalsequences are heterogeneous and differ greatly in their primary aminoacid sequences. However, signal sequences are typically 16 to 30 aminoacids in length and include a hydrophilic, usually positively chargedN-terminal region, a central hydrophobic domain, and a C-terminal regionthat contains the cleavage site for signal peptidase.

In some embodiments, a single-chain chimeric polypeptide includes asignal sequence having an amino acid sequence MKWVTFISLLFLFSSAYS (SEQ IDNO: 62). In some embodiments, a single chain chimeric polypeptideincludes a signal sequence encoded by the nucleic acid sequence

(SEQ ID NO: 63) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTA CTCC,(SEQ ID NO: 64) ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTA CAGC,or (SEQ ID NO: 65) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC.

In some embodiments, a single-chain chimeric polypeptide includes asignal sequence having an amino acid sequence MKCLLYLAFLFLGVNC (SEQ IDNO: 66). In some embodiments, a single-chain chimeric polypeptideincludes a signal sequence having an amino acid sequenceMGQIVTMFEALPHIIDEVINMIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG (SEQ ID NO:67). In some embodiments, a single-chain chimeric polypeptide includes asignal sequence having an amino acid sequenceMPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO: 68). In someembodiments, a single-chain chimeric polypeptide includes a signalsequence having an amino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS(SEQ ID NO: 69). Those of ordinary skill in the art will be aware ofother appropriate signal sequences for use in a single-chain chimericpolypeptide.

In some embodiments, a single-chain chimeric polypeptide includes asignal sequence that is about 10 to 100 amino acids in length. Forexample, a signal sequence can be about 10 to 100 amino acids in length,about 15 to 100 amino acids in length, about 20 to 100 amino acids inlength, about 25 to 100 amino acids in length, about 30 to 100 aminoacids in length, about 35 to 100 amino acids in length, about 40 to 100amino acids in length, about 45 to 100 amino acids in length, about 50to 100 amino acids in length, about 55 to 100 amino acids in length,about 60 to 100 amino acids in length, about 65 to 100 amino acids inlength, about 70 to 100 amino acids in length, about 75 to 100 aminoacids in length, about 80 to 100 amino acids in length, about 85 to 100amino acids in length, about 90 to 100 amino acids in length, about 95to 100 amino acids in length, about 10 to 95 amino acids in length,about 10 to 90 amino acids in length, about 10 to 85 amino acids inlength, about 10 to 80 amino acids in length, about 10 to 75 amino acidsin length, about 10 to 70 amino acids in length, about 10 to 65 aminoacids in length, about 10 to 60 amino acids in length, about 10 to 55amino acids in length, about 10 to 50 amino acids in length, about 10 to45 amino acids in length, about 10 to 40 amino acids in length, about 10to 35 amino acids in length, about 10 to 30 amino acids in length, about10 to 25 amino acids in length, about 10 to 20 amino acids in length,about 10 to 15 amino acids in length, about 20 to 30 amino acids inlength, about 30 to 40 amino acids in length, about 40 to 50 amino acidsin length, about 50 to 60 amino acids in length, about 60 to 70 aminoacids in length, about 70 to 80 amino acids in length, about 80 to 90amino acids in length, about 90 to 100 amino acids in length, about 20to 90 amino acids in length, about 30 to 80 amino acids in length, about40 to 70 amino acids in length, about 50 to 60 amino acids in length, orany range in between. In some embodiments, a signal sequence is about10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 amino acids in length.

In some embodiments, any of the signal sequences disclosed herein caninclude one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8,9, 10, or more amino acids) at its N-terminus and/or C-terminus, so longas the function of the signal sequence remains intact. For example, asignal sequence having the amino acid sequence MKWVTFISLLFLFSSAYS (SEQID NO: 62) can include one or more additional amino acids at theN-terminus or C-terminus, while still retaining the ability to directthe single-chain chimeric polypeptide to the secretory pathway.

In some embodiments, a single-chain chimeric polypeptide includes asignal sequence that directs the single-chain chimeric polypeptide intothe extracellular space. Such embodiments are useful in producingsingle-chain chimeric polypeptides that are relatively easy to beisolated and/or purified.

Peptide Tags

In some embodiments, a single-chain chimeric polypeptide includes apeptide tag (e.g., at the N-terminal end or the C-terminal end of thesingle-chain chimeric polypeptide). In some embodiments, a single-chainchimeric polypeptide includes two or more peptide tags.

Exemplary peptide tags that can be included in a single-chain chimericpolypeptide include, without limitation, AviTag (GLNDIFEAQKIEWHE; SEQ IDNO: 70), a calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 71), apolyglutamate tag (EEEEEE; SEQ ID NO: 72), an E-tag (GAPVPYPDPLEPR; SEQID NO: 73), a FLAG-tag (DYKDDDDK; SEQ ID NO: 74), an HA-tag, a peptidefrom hemagglutinin (YPYDVPDYA; SEQ ID NO: 75), a his-tag (HHHHH (SEQ IDNO: 76); HHHHHH (SEQ ID NO: 77); HHHHHHH (SEQ ID NO: 78); HHHHHHHH (SEQID NO: 79); HHHHHHHHH (SEQ ID NO: 80); or HHHHHHHHHH (SEQ ID NO: 81)), amyc-tag (EQKLISEEDL; SEQ ID NO: 82), NE-tag (TKENPRSNQEESYDDNES; SEQ IDNO: 83), S-tag, (KETAAAKFERQHMDS; SEQ ID NO: 84), SBP-tag(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 85), Softag 1(SLAELLNAGLGGS; SEQ ID NO: 86), Softag 3 (TQDPSRVG; SEQ ID NO: 87),Spot-tag (PDRVRAVSHWSS; SEQ ID NO: 88), Strep-tag (WSHPQFEK; SEQ ID NO:89), TC tag (CCPGCC; SEQ ID NO: 90), Ty tag (EVHTNQDPLD; SEQ ID NO: 91),V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 92), VSV-tag (YTDIEMNRLGK; SEQ ID NO:93), and Xpress tag (DLYDDDDK; SEQ ID NO: 94). In some embodiments,tissue factor protein is a peptide tag.

Peptide tags that can be included in a single-chain chimeric polypeptidecan be used in any of a variety of applications related to thesingle-chain chimeric polypeptide. For example, a peptide tag can beused in the purification of a single-chain chimeric polypeptide. As onenon-limiting example, a single-chain chimeric polypeptide can include amyc tag; and can be purified using an antibody that recognizes the myctag(s). One non-limiting example of an antibody that recognizes a myctag is 9E10, available from the non-commercial Developmental StudiesHybridoma Bank. As another non-limiting example, a single-chain chimericpolypeptide can include a histidine tag, and can be purified using anickel or cobalt chelate. Those of ordinary skill in the art will beaware of other suitable tags and agent that bind those tags for use inpurifying a single-chain chimeric polypeptide. In some embodiments, apeptide tag is removed from the single-chain chimeric polypeptide afterpurification. In some embodiments, a peptide tag is not removed from thesingle-chain chimeric polypeptide after purification.

Peptide tags that can be included in a single-chain chimeric polypeptidecan be used, for example, in immunoprecipitation of the single-chainchimeric polypeptide, imaging of the single-chain chimeric polypeptide(e.g., via Western blotting, ELISA, flow cytometry, and/orimmunocytochemistry), and/or solubilization of the single-chain chimericpolypeptide.

In some embodiments, a single-chain chimeric polypeptide includes apeptide tag that is about 10 to 100 amino acids in length. For example,a peptide tag can be about 10 to 100 amino acids in length, about 15 to100 amino acids in length, about 20 to 100 amino acids in length, about25 to 100 amino acids in length, about 30 to 100 amino acids in length,about 35 to 100 amino acids in length, about 40 to 100 amino acids inlength, about 45 to 100 amino acids in length, about 50 to 100 aminoacids in length, about 55 to 100 amino acids in length, about 60 to 100amino acids in length, about 65 to 100 amino acids in length, about 70to 100 amino acids in length, about 75 to 100 amino acids in length,about 80 to 100 amino acids in length, about 85 to 100 amino acids inlength, about 90 to 100 amino acids in length, about 95 to 100 aminoacids in length, about 10 to 95 amino acids in length, about 10 to 90amino acids in length, about 10 to 85 amino acids in length, about 10 to80 amino acids in length, about 10 to 75 amino acids in length, about 10to 70 amino acids in length, about 10 to 65 amino acids in length, about10 to 60 amino acids in length, about 10 to 55 amino acids in length,about 10 to 50 amino acids in length, about 10 to 45 amino acids inlength, about 10 to 40 amino acids in length, about 10 to 35 amino acidsin length, about 10 to 30 amino acids in length, about 10 to 25 aminoacids in length, about 10 to 20 amino acids in length, about 10 to 15amino acids in length, about 20 to 30 amino acids in length, about 30 to40 amino acids in length, about 40 to 50 amino acids in length, about 50to 60 amino acids in length, about 60 to 70 amino acids in length, about70 to 80 amino acids in length, about 80 to 90 amino acids in length,about 90 to 100 amino acids in length, about 20 to 90 amino acids inlength, about 30 to 80 amino acids in length, about 40 to 70 amino acidsin length, about 50 to 60 amino acids in length, or any range inbetween. In some embodiments, a peptide tag is about 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidsin length.

Peptide tags included in a single-chain chimeric polypeptide can be ofany suitable length. For example, peptide tags can be 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length.In embodiments in which a single-chain chimeric polypeptide includes twoor more peptide tags, the two or more peptide tags can be of the same ordifferent lengths. In some embodiments, any of the peptide tagsdisclosed herein may include one or more additional amino acids (e.g.,1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at the N-terminusand/or C-terminus, so long as the function of the peptide tag remainsintact. For example, a myc tag having the amino acid sequence EQKLISEEDL(SEQ ID NO: 82) can include one or more additional amino acids (e.g., atthe N-terminus and/or the C-terminus of the peptide tag), while stillretaining the ability to be bound by an antibody (e.g., 9E10).

Exemplary Embodiments of Single-Chain Chimeric Polypeptides—Type A

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain and/or the secondtarget-binding domain can independently bind specifically to CD3 (e.g.,human CD3) or CD28 (e.g., human CD28). In some embodiments, the firsttarget-binding domain binds specifically to CD3 (e.g., human CD3) andthe second target-binding domain binds specifically to CD28 (e.g., humanCD28). In some embodiments, the first target-binding domain bindsspecifically to CD28 (e.g., human CD28) and the second target-bindingdomain binds specifically to CD3 (e.g., human CD3).

In some embodiments of these single-chain chimeric polypeptides, thefirst target-binding domain and the soluble tissue factor domaindirectly abut each other. In some embodiments of these single-chainchimeric polypeptides, the single-chain chimeric polypeptide furtherincludes a linker sequence (e.g., any of the exemplary linkers describedherein) between the first target-binding domain and the soluble tissuefactor domain.

In some embodiments of these single-chain chimeric polypeptides, thesoluble tissue factor domain and the second target-binding domaindirectly abut each other. In some embodiments of these single-chainchimeric polypeptides, the single-chain chimeric polypeptide furtherincludes a linker sequence (e.g., any of the exemplary linkers describedherein) between the soluble tissue factor domain and the secondtarget-binding domain.

In some embodiments of these single-chain chimeric polypeptides, one orboth of the first target-binding domain and the second target-bindingdomain is an antigen-binding domain. In some embodiments of thesesingle-chain chimeric polypeptides, the first target-binding domain andthe second target-binding domain are each an antigen-binding domain(e.g., any of the exemplary antigen-binding domains described herein).In some embodiments of these single-chain chimeric polypeptides, theantigen-binding domain includes a scFv or a single domain antibody.

A non-limiting example of an scFv that binds specifically to CD3 caninclude a sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 20) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS.

In some embodiments, an scFv that binds specifically to CD3 can beencoded by a sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 99) CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGC.

A non-limiting example of an scFv that binds specifically to CD28 caninclude a sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 26) VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR.

In some embodiments, an scFv that binds specifically to CD28 can beencoded by a sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 101) GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG.

In some embodiments of these single-chain chimeric polypeptides, thefirst target-binding domain and/or the second target-binding domain is asoluble receptor (e.g., a soluble CD28 receptor or a soluble CD3receptor). In some embodiments of these single-chain chimericpolypeptides, the soluble tissue factor domain can be any of theexemplary soluble tissue factor domains described herein.

In some embodiments, a single-chain chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 1) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTF GGGTKLETKR.

In some embodiments, a single-chain chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 2) CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG.

In some embodiments, a single-chain chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 3) MKWVTFISLLFLFSSAYSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGS SPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHR SPTFGGGTKLETKR.

In some embodiments, a single-chain chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 4) ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCTATTCCCAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG.

Exemplary Embodiments of Single-Chain Chimeric Polypeptides—Type B

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain and/or the secondtarget-binding domain can independently bind specifically to an IL-2receptor (e.g., human IL-2 receptor).

In some embodiments of these single-chain chimeric polypeptides, thefirst target-binding domain and the soluble tissue factor domaindirectly abut each other. In some embodiments of these single-chainchimeric polypeptides, the single-chain chimeric polypeptide furtherincludes a linker sequence (e.g., any of the exemplary linkers describedherein) between the first target-binding domain and the soluble tissuefactor domain.

In some embodiments of these single-chain chimeric polypeptides, thesoluble tissue factor domain and the second target-binding domaindirectly abut each other. In some embodiments of these single-chainchimeric polypeptides, the single-chain chimeric polypeptide furtherincludes a linker sequence (e.g., any of the exemplary linkers describedherein) between the soluble tissue factor domain and the secondtarget-binding domain.

In some embodiments of these single-chain chimeric polypeptides, thefirst target-binding domain and the second target-binding domain is asoluble human IL-2 protein. A non-limiting example of an IL-2 proteinthat binds specifically to an IL-2 receptor can include a sequence thatis at least 80% identical (e.g., at least 82% identical, at least 84%identical, at least 86% identical, at least 88% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 28) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

In some embodiments, an IL-2 protein that binds specifically to an IL-2receptor can be encoded by a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 106) GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAAC ACTAACT.

In some embodiments, an IL-2 protein that binds specifically to an IL-2receptor can be encoded by a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 107) GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATC ATCTCCACTTTAACC

In some embodiments of these single-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein.

In some embodiments, a single-chain chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 108) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

In some embodiments, a single-chain chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 109) GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACCAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT.

In some embodiments, a single-chain chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 110) MKWVTFISLLFLFSSAYSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT.

In some embodiments, a single-chain chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 111) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACCAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAA AGCATCATCTCAACACTAACT.

Exemplary Embodiments of Single-Chain Chimeric Polypeptides—Type C

In some embodiments of any of the single-chain chimeric polypeptidesdescribed herein, the first target-binding domain and/or the secondtarget-binding domain can independently bind specifically to an IL-15receptor (e.g., a human IL-15 receptor).

In some embodiments of these single-chain chimeric polypeptides, thefirst target-binding domain and the soluble tissue factor domaindirectly abut each other. In some embodiments of these single-chainchimeric polypeptides, the single-chain chimeric polypeptide furtherincludes a linker sequence (e.g., any of the exemplary linkers describedherein) between the first target-binding domain and the soluble tissuefactor domain.

In some embodiments of these single-chain chimeric polypeptides, thesoluble tissue factor domain and the second target-binding domaindirectly abut each other. In some embodiments of these single-chainchimeric polypeptides, the single-chain chimeric polypeptide furtherincludes a linker sequence (e.g., any of the exemplary linkers describedherein) between the soluble tissue factor domain and the secondtarget-binding domain.

In some embodiments of these single-chain chimeric polypeptides, thefirst target-binding domain and the second target-binding domain is asoluble human IL-15 protein. A non-limiting example of an IL-15 proteinthat binds specifically to an IL-15 receptor can include a sequence thatis at least 80% identical (e.g., at least 82% identical, at least 84%identical, at least 86% identical, at least 88% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 39) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKE FLQSFVHIVQMFINTS.

In some embodiments, an IL-15 protein that binds specifically to anIL-15 receptor can be encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 112) AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAAC ACTAGC.

In some embodiments, an IL-15 protein that binds specifically to anIL-15 receptor can be encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 113) AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC 

In some embodiments of these single-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein.

In some embodiments, a single-chain chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 114) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS.

In some embodiments, a single-chain chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 115) AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTAGCAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAG ATGTTCATCAATACCTCC.

In some embodiments, a single-chain chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 116) MKWVTFISLLFLFSSAYSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a single-chain chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 117) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCAACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTAGCAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC.

Compositions/Kits

Also provided herein are compositions (e.g., pharmaceuticalcompositions) that include at least one of any single-chain chimericpolypeptides, any of the cells, or any of the nucleic acids describedherein. In some embodiments, the compositions include at least one ofany of the single-chain chimeric polypeptides described herein. In someembodiments, the compositions include any of the immune cells (e.g., anyof the immune cells described herein, e.g., any of the immune cellsproduced using any of the methods described herein).

In some embodiments, the pharmaceutical compositions are formulated fordifferent routes of administration (e.g., intravenous, subcutaneous). Insome embodiments, the pharmaceutical compositions can include apharmaceutically acceptable carrier (e.g., phosphate buffered saline).

Single or multiple administrations of pharmaceutical compositions can begiven to a subject in need thereof depending on for example: the dosageand frequency as required and tolerated by the subject. The formulationshould provide a sufficient quantity of active agent to effectivelytreat, prevent or ameliorate conditions, diseases or symptoms.

Also provided herein are kits that include any of the single-chainchimeric polypeptides, compositions, nucleic acids, or cells (e.g.,immune cells) described herein. In some embodiments, the kits caninclude instructions for performing any of the methods described herein.In some embodiments, the kits can include at least one dose of any ofthe pharmaceutical compositions described herein.

Nucleic Acids/Vectors

Also provided herein are nucleic acids that encode any of thesingle-chain chimeric polypeptides described herein. Also providedherein are vectors that include any of the nucleic acids encoding any ofthe single-chain chimeric polypeptides described herein.

Any of the vectors described herein can be an expression vector. Forexample, an expression vector can include a promoter sequence operablylinked to the sequence encoding the single-chain chimeric polypeptide.

Non-limiting examples of vectors include plasmids, transposons, cosmids,and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMVvectors), adeno-associated virus (AAV) vectors, lentivirus vectors, andretroviral vectors), and any Gateway® vectors. A vector can, e.g.,include sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host mammalian cell or in an invitro expression system. Skilled practitioners will be capable ofselecting suitable vectors and mammalian cells for making any of thesingle-chain chimeric polypeptides described herein.

Cells

Also provided herein are cells (e.g., any of the exemplary cellsdescribed herein or known in the art) comprising any of the nucleicacids described herein that encode any of the single-chain chimericpolypeptides described herein.

Also provided herein are cells (e.g., any of the exemplary cellsdescribed herein or known in the art) that include any of the vectorsdescribed herein that encode any of the single-chain chimericpolypeptides described herein.

In some embodiments of any of the methods described herein, the cell canbe a eukaryotic cell. As used herein, the term “eukaryotic cell” refersto a cell having a distinct, membrane-bound nucleus. Such cells mayinclude, for example, mammalian (e.g., rodent, non-human primate, orhuman), insect, fungal, or plant cells. In some embodiments, theeukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. Insome embodiments, the eukaryotic cell is a higher eukaryote, such asmammalian, avian, plant, or insect cells. Non-limiting examples ofmammalian cells include Chinese hamster ovary cells and human embryonickidney cells (e.g., HEK293 cells).

Methods of introducing nucleic acids and expression vectors into a cell(e.g., an eukaryotic cell) are known in the art. Non-limiting examplesof methods that can be used to introduce a nucleic acid into a cellinclude lipofection, transfection, electroporation, microinjection,calcium phosphate transfection, dendrimer-based transfection, cationicpolymer transfection, cell squeezing, sonoporation, opticaltransfection, impalefection, hydrodynamic delivery, magnetofection,viral transduction (e.g., adenoviral and lentiviral transduction), andnanoparticle transfection.

Methods of Producing Single-Chain Chimeric Polypeptides

Also provided herein are methods of producing any of the single-chainchimeric polypeptides described herein that include culturing any of thecells described herein in a culture medium under conditions sufficientto result in the production of the single-chain chimeric polypeptide;and recovering the single-chain chimeric polypeptide from the celland/or the culture medium.

The recovery of the single-chain chimeric polypeptide from a culturemedium or a cell (e.g., a eukaryotic cell) can be performed usingtechniques well-known in the art (e.g., ammonium sulfate precipitation,polyethylene glycol precipitation, ion-exchange chromatography (anion orcation), chromatography based on hydrophobic interaction, metal-affinitychromatography, ligand-affinity chromatography, and size exclusionchromatography).

Methods of culturing cells are well known in the art. Cells can bemaintained in vitro under conditions that favor proliferation,differentiation and growth. Briefly, cells can be cultured by contactinga cell (e.g., any cell) with a cell culture medium that includes thenecessary growth factors and supplements to support cell viability andgrowth.

Also provided herein are single-chain chimeric polypeptides (e.g., anyof the single-chain chimeric polypeptides described herein) produced byany of the methods described herein.

Methods of Stimulating an Immune Cell

Also provided herein are methods of stimulating an immune cell (e.g.,any of the exemplary immune cells described herein or known in the art)that include contacting an immune cell with an effective amount of anyof the single-chain chimeric polypeptides described herein or any of thecompositions (e.g., pharmaceutical compositions) described herein. Insome examples, the immune cell is contacted in vitro (e.g., in asuitable liquid culture medium under conditions sufficient to result instimulation of the immune cell).

In some examples, the immune cell has been previously obtained from asubject (e.g., a mammal, e.g., a human). Some embodiments of thesemethods further include obtaining the immune cell from the subject priorto the contacting step.

In some examples, the immune cell is contacted in vivo. In suchembodiments, the single-chain chimeric polypeptide is administered to asubject (e.g., a mammal, e.g., a human) in an amount sufficient toresult in stimulation of an immune cell in the subject.

In some examples of any of the methods described herein, the immune cellcan be an immature thymocyte, a peripheral blood lymphocyte, a naïve Tcell, a pluripotent Th cell precursor, a lymphoid progenitor cell, aTreg cell, a memory T cell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2cell, a Th1 cell, a Th3 cell, γδ T cell, an αβ T cell, atumor-infiltrating T cell, a CD8⁺ T cell, a CD4⁺ T cell, a naturalkiller T cell, a mast cell, a macrophage, a neutrophil, a dendriticcell, a basophil, an eosinophil, or a natural killer cell, or acombination thereof.

In some examples, the immune cell has previously beengenetically-modified to express a chimeric antigen receptor or arecombinant T-cell receptor. In some examples, the immune cell (e.g.,any of the immune cells described herein) has previously beengenetically-modified to express a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include, after thecontacting step, introducing into the immune cell (e.g., any of theimmune cells described herein) a nucleic acid encoding a chimericantigen-receptor or a recombinant T-cell receptor. Some embodiments ofthese methods can further include, after the contacting step,introducing into the immune cell (e.g., any of the immune cellsdescribed herein) a nucleic acid encoding a co-stimulatory molecule(e.g., CD28).

Some embodiments of these methods can further include administering atherapeutically effective amount of the immune cell to a subject in needthereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosedas having an age-related disease or condition. Non-limiting examples ofage-related diseases or disorders include: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identifiedor diagnosed as having a cancer. Non-limiting examples of cancersinclude: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosedor identified as having an infectious disease. Non-limiting examples ofinfectious disease include infection with human immunodeficiency virus,cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus,smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus,and hepatitis C virus, papillomavirus, or influenza virus.

Activation of an immune cell can be determined using methods known inthe art. For example, activation of an immune cell can be determined bydetecting the levels of cytokines and chemokines that are secreted uponactivation of an immune cell. Non-limiting examples of cytokines,chemokines, and regulatory molecules that are secreted or upregulatedupon activation of an immune cell include: IL-2, IFN-γ, IL-1, IL-4,IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22,IL-33, leukotriene B4, CCL5, TNFα, granzymes, perforin, TGFβ, STAT3,RORγT, FOXP3, STATE, and GATA3. The detection of these cytokines andchemokines or regulatory molecules can be performed using an immunoassay(e.g., an enzyme-linked immunosorbent assay) or quantitative PCR. Forexample, activation of an immune cell can result in an increase of about1% to about 800% (e.g., about 1% to about 750%, about 1% to about 700%,about 1% to about 650%, about 1% to about 600%, about 1% to about 550%,about 1% to about 500%, about 1% to about 450%, about 1% to about 400%,about 1% to about 350%, about 1% to about 300%, about 1% to about 280%,about 1% to about 260%, about 1% to about 240%, about 1% to about 220%,about 1% to about 200%, about 1% to about 180%, about 1% to about 160%,about 1% to about 140%, about 1% to about 120%, about 1% to about 100%,about 1% to about 90%, about 1% to about 80%, about 1% to about 70%,about 1% to about 60%, about 1% to about 50%, about 1% to about 45%,about 1% to about 40%, about 1% to about 35%, about 1% to about 30%,about 1% to about 25%, about 1% to about 20%, about 1% to about 15%,about 1% to about 10%, about 1% to about 5%, about 5% to about 800%,about 5% to about 750%, about 5% to about 700%, about 5% to about 650%,about 5% to about 600%, about 5% to about 550%, about 5% to about 500%,about 5% to about 450%, about 5% to about 400%, about 5% to about 350%,about 5% to about 300%, about 5% to about 280%, about 5% to about 260%,about 5% to about 240%, about 5% to about 220%, about 5% to about 200%,about 5% to about 180%, about 5% to about 160%, about 5% to about 140%,about 5% to about 120%, about 5% to about 100%, about 5% to about 90%,about 5% to about 80%, about 5% to about 70%, about 5% to about 60%,about 5% to about 50%, about 5% to about 45%, about 5% to about 40%,about 5% to about 35%, about 5% to about 30%, about 5% to about 25%,about 5% to about 20%, about 5% to about 15%, about 5% to about 10%,about 10% to about 800%, about 10% to about 750%, about 10% to about700%, about 10% to about 650%, about 10% to about 600%, about 10% toabout 550%, about 10% to about 500%, about 10% to about 450%, about 10%to about 400%, about 10% to about 350%, about 10% to about 300%, about10% to about 280%, about 10% to about 260%, about 10% to about 240%,about 10% to about 220%, about 10% to about 200%, about 10% to about180%, about 10% to about 160%, about 10% to about 140%, about 10% toabout 120%, about 10% to about 100%, about 10% to about 90%, about 10%to about 80%, about 10% to about 70%, about 10% to about 60%, about 10%to about 50%, about 10% to about 45%, about 10% to about 40%, about 10%to about 35%, about 10% to about 30%, about 10% to about 25%, about 10%to about 20%, about 10% to about 15%, about 15% to about 800%, about 15%to about 750%, about 15% to about 700%, about 15% to about 650%, about15% to about 600%, about 15% to about 550%, about 15% to about 500%,about 15% to about 450%, about 15% to about 400%, about 15% to about350%, about 15% to about 300%, about 15% to about 280%, about 15% toabout 260%, about 15% to about 240%, about 15% to about 220%, about 15%to about 200%, about 15% to about 180%, about 15% to about 160%, about15% to about 140%, about 15% to about 120%, about 15% to about 100%,about 15% to about 90%, about 15% to about 80%, about 15% to about 70%,about 15% to about 60%, about 15% to about 50%, about 15% to about 45%,about 15% to about 40%, about 15% to about 35%, about 15% to about 30%,about 15% to about 25%, about 15% to about 20%, about 20% to about 800%,about 20% to about 750%, about 20% to about 700%, about 20% to about650%, about 20% to about 600%, about 20% to about 550%, about 20% toabout 500%, about 20% to about 450%, about 20% to about 400%, about 20%to about 350%, about 20% to about 300%, about 20% to about 280%, about20% to about 260%, about 20% to about 240%, about 20% to about 220%,about 20% to about 200%, about 20% to about 180%, about 20% to about160%, about 20% to about 140%, about 20% to about 120%, about 20% toabout 100%, about 20% to about 90%, about 20% to about 80%, about 20% toabout 70%, about 20% to about 60%, about 20% to about 50%, about 20% toabout 45%, about 20% to about 40%, about 20% to about 35%, about 20% toabout 30%, about 20% to about 25%, about 25% to about 800%, about 25% toabout 750%, about 25% to about 700%, about 25% to about 650%, about 25%to about 600%, about 25% to about 550%, about 25% to about 500%, about25% to about 450%, about 25% to about 400%, about 25% to about 350%,about 25% to about 300%, about 25% to about 280%, about 25% to about260%, about 25% to about 240%, about 25% to about 220%, about 25% toabout 200%, about 25% to about 180%, about 25% to about 160%, about 25%to about 140%, about 25% to about 120%, about 25% to about 100%, about25% to about 90%, about 25% to about 80%, about 25% to about 70%, about25% to about 60%, about 25% to about 50%, about 25% to about 45%, about25% to about 40%, about 25% to about 35%, about 35% to about 800%, about35% to about 750%, about 35% to about 700%, about 35% to about 650%,about 35% to about 600%, about 35% to about 550%, about 35% to about500%, about 35% to about 450%, about 35% to about 400%, about 35% toabout 350%, about 35% to about 300%, about 35% to about 280%, about 35%to about 260%, about 35% to about 240%, about 35% to about 220%, about35% to about 200%, about 35% to about 180%, about 35% to about 160%,about 35% to about 140%, about 35% to about 120%, about 35% to about100%, about 35% to about 90%, about 35% to about 80%, about 35% to about70%, about 35% to about 60%, about 35% to about 50%, about 35% to about45%, about 35% to about 40%, about 40% to about 800%, about 40% to about750%, about 40% to about 700%, about 40% to about 650%, about 40% toabout 600%, about 40% to about 550%, about 40% to about 500%, about 40%to about 450%, about 40% to about 400%, about 40% to about 350%, about40% to about 300%, about 40% to about 280%, about 40% to about 260%,about 40% to about 240%, about 40% to about 220%, about 40% to about200%, about 40% to about 180%, about 40% to about 160%, about 40% toabout 140%, about 40% to about 120%, about 40% to about 100%, about 40%to about 90%, about 40% to about 80%, about 40% to about 70%, about 40%to about 60%, about 40% to about 50%, about 40% to about 45%, about 45%to about 800%, about 45% to about 750%, about 45% to about 700%, about45% to about 650%, about 45% to about 600%, about 45% to about 550%,about 45% to about 500%, about 45% to about 450%, about 45% to about400%, about 45% to about 350%, about 45% to about 300%, about 45% toabout 280%, about 45% to about 260%, about 45% to about 240%, about 45%to about 220%, about 45% to about 200%, about 45% to about 180%, about45% to about 160%, about 45% to about 140%, about 45% to about 120%,about 45% to about 100%, about 45% to about 90%, about 45% to about 80%,about 45% to about 70%, about 45% to about 60%, about 45% to about 50%,about 50% to about 800%, about 50% to about 750%, about 50% to about700%, about 50% to about 650%, about 50% to about 600%, about 50% toabout 550%, about 50% to about 500%, about 50% to about 450%, about 50%to about 400%, about 50% to about 350%, about 50% to about 300%, about50% to about 280%, about 50% to about 260%, about 50% to about 240%,about 50% to about 220%, about 50% to about 200%, about 50% to about180%, about 50% to about 160%, about 50% to about 140%, about 50% toabout 120%, about 50% to about 100%, about 50% to about 90%, about 50%to about 80%, about 50% to about 70%, about 50% to about 60%, about 60%to about 800%, about 60% to about 750%, about 60% to about 700%, about60% to about 650%, about 60% to about 600%, about 60% to about 550%,about 60% to about 500%, about 60% to about 450%, about 60% to about400%, about 60% to about 350%, about 60% to about 300%, about 60% toabout 280%, about 60% to about 260%, about 60% to about 240%, about 60%to about 220%, about 60% to about 200%, about 60% to about 180%, about60% to about 160%, about 60% to about 140%, about 60% to about 120%,about 60% to about 100%, about 60% to about 90%, about 60% to about 80%,about 60% to about 70%, about 70% to about 800%, about 70% to about750%, about 70% to about 700%, about 70% to about 650%, about 70% toabout 600%, about 70% to about 550%, about 70% to about 500%, about 70%to about 450%, about 70% to about 400%, about 70% to about 350%, about70% to about 300%, about 70% to about 280%, about 70% to about 260%,about 70% to about 240%, about 70% to about 220%, about 70% to about200%, about 70% to about 180%, about 70% to about 160%, about 70% toabout 140%, about 70% to about 120%, about 70% to about 100%, about 70%to about 90%, about 70% to about 80%, about 80% to about 800%, about 80%to about 750%, about 80% to about 700%, about 80% to about 650%, about80% to about 600%, about 80% to about 550%, about 80% to about 500%,about 80% to about 450%, about 80% to about 400%, about 80% to about350%, about 80% to about 300%, about 80% to about 280%, about 80% toabout 260%, about 80% to about 240%, about 80% to about 220%, about 80%to about 200%, about 80% to about 180%, about 80% to about 160%, about80% to about 140%, about 80% to about 120%, about 80% to about 100%,about 80% to about 90%, about 90% to about 800%, about 90% to about750%, about 90% to about 700%, about 90% to about 650%, about 90% toabout 600%, about 90% to about 550%, about 90% to about 500%, about 90%to about 450%, about 90% to about 400%, about 90% to about 350%, about90% to about 300%, about 90% to about 280%, about 90% to about 260%,about 90% to about 240%, about 90% to about 220%, about 90% to about200%, about 90% to about 180%, about 90% to about 160%, about 90% toabout 140%, about 90% to about 120%, about 90% to about 100%, about 100%to about 800%, about 100% to about 750%, about 100% to about 700%, about100% to about 650%, about 100% to about 600%, about 100% to about 550%,about 100% to about 500%, about 100% to about 450%, about 100% to about400%, about 100% to about 350%, about 100% to about 300%, about 100% toabout 280%, about 100% to about 260%, about 100% to about 240%, about100% to about 220%, about 100% to about 200%, about 100% to about 180%,about 100% to about 160%, about 100% to about 140%, about 100% to about120%, about 120% to about 800%, about 120% to about 750%, about 120% toabout 700%, about 120% to about 650%, about 120% to about 600%, about120% to about 550%, about 120% to about 500%, about 120% to about 450%,about 120% to about 400%, about 120% to about 350%, about 120% to about300%, about 120% to about 280%, about 120% to about 260%, about 120% toabout 240%, about 120% to about 220%, about 120% to about 200%, about120% to about 180%, about 120% to about 160%, about 120% to about 140%,about 140% to about 800%, about 140% to about 750%, about 140% to about700%, about 140% to about 650%, about 140% to about 600%, about 140% toabout 550%, about 140% to about 500%, about 140% to about 450%, about140% to about 400%, about 140% to about 350%, about 140% to about 300%,about 140% to about 280%, about 140% to about 260%, about 140% to about240%, about 140% to about 220%, about 140% to about 200%, about 140% toabout 180%, about 140% to about 160%, about 160% to about 800%, about160% to about 750%, about 160% to about 700%, about 160% to about 650%,about 160% to about 600%, about 160% to about 550%, about 160% to about500%, about 160% to about 450%, about 160% to about 400%, about 160% toabout 350%, about 160% to about 300%, about 160% to about 280%, about160% to about 260%, about 160% to about 240%, about 160% to about 220%,about 160% to about 200%, about 160% to about 180%, about 180% to about800%, about 180% to about 750%, about 180% to about 700%, about 180% toabout 650%, about 180% to about 600%, about 180% to about 550%, about180% to about 500%, about 180% to about 450%, about 180% to about 400%,about 180% to about 350%, about 180% to about 300%, about 180% to about280%, about 180% to about 260%, about 180% to about 240%, about 180% toabout 220%, about 180% to about 200%, about 200% to about 800%, about200% to about 750%, about 200% to about 700%, about 200% to about 650%,about 200% to about 600%, about 200% to about 550%, about 200% to about500%, about 200% to about 450%, about 200% to about 400%, about 200% toabout 350%, about 200% to about 300%, about 200% to about 280%, about200% to about 260%, about 200% to about 240%, about 200% to about 220%,about 220% to about 800%, about 220% to about 750%, about 220% to about700%, about 220% to about 650%, about 220% to about 600%, about 220% toabout 550%, about 220% to about 500%, about 220% to about 450%, about220% to about 400%, about 220% to about 350%, about 220% to about 300%,about 220% to about 280%, about 220% to about 260%, about 220% to about240%, about 240% to about 800%, about 240% to about 750%, about 240% toabout 700%, about 240% to about 650%, about 240% to about 600%, about240% to about 550%, about 240% to about 500%, about 240% to about 450%,about 240% to about 400%, about 240% to about 350%, about 240% to about300%, about 240% to about 280%, about 240% to about 260%, about 260% toabout 800%, about 260% to about 750%, about 260% to about 700%, about260% to about 650%, about 260% to about 600%, about 260% to about 550%,about 260% to about 500%, about 260% to about 450%, about 260% to about400%, about 260% to about 350%, about 260% to about 300%, about 260% toabout 280%, about 280% to about 800%, about 280% to about 750%, about280% to about 700%, about 280% to about 650%, about 280% to about 600%,about 280% to about 550%, about 280% to about 500%, about 280% to about450%, about 280% to about 400%, about 280% to about 350%, about 280% toabout 300%, about 300% to about 800%, about 300% to about 750%, about300% to about 700%, about 300% to about 650%, about 300% to about 600%,about 300% to about 550%, about 300% to about 500%, about 300% to about450%, about 300% to about 400%, about 300% to about 350%, about 350% toabout 800%, about 350% to about 750%, about 350% to about 700%, about350% to about 650%, about 350% to about 600%, about 350% to about 550%,about 350% to about 500%, about 350% to about 450%, about 350% to about400%, about 400% to about 800%, about 400% to about 750%, about 400% toabout 700%, about 400% to about 650%, about 400% to about 600%, about400% to about 550%, about 400% to about 500%, about 400% to about 450%,about 450% to about 800%, about 450% to about 750%, about 450% to about700%, about 450% to about 650%, about 450% to about 600%, about 450% toabout 550%, about 450% to about 500%, about 500% to about 800%, about500% to about 750%, about 500% to about 700%, about 500% to about 650%,about 500% to about 600%, about 500% to about 550%, about 550% to about800%, about 550% to about 750%, about 550% to about 700%, about 550% toabout 650%, about 550% to about 600%, about 600% to about 800%, about600% to about 750%, about 600% to about 700%, about 600% to about 650%,about 650% to about 800%, about 650% to about 750%, about 650% to about700%, about 700% to about 800%, about 700% to about 750%, or about 750%to about 800%) of one or more of any of the cytokines or chemokines orregulatory molecules described herein (e.g., one or more of any of IL-2,IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15,IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFα, granzymes,perforin, TGFβ, STAT3, RORγT, FOXP3, STATE, and GATA3) (e.g., ascompared to the level of the one or more cytokines and chemokines in acontrol not contacted with any of the single-chain chimeric polypeptidesdescribed herein).

Methods of Inducing or Increasing Proliferation of an Immune Cell

Also provided herein are methods of inducing or increasing proliferationof an immune cell (e.g., any of the exemplary immune cells describedherein or known in the art) that include contacting an immune cell withan effective amount of any of the single-chain chimeric polypeptidesdescribed herein or any of the compositions (e.g., pharmaceuticalcompositions) described herein. In some examples, the immune cell iscontacted in vitro (e.g., in a suitable liquid culture medium underconditions sufficient to result in stimulation of the immune cell).

In some examples, the immune cell has been previously obtained from asubject (e.g., a mammal, e.g., a human). Some embodiments of thesemethods further include obtaining the immune cell from the subject priorto the contacting step.

In some examples, the immune cell is contacted in vivo. In suchembodiments, the single-chain chimeric polypeptide is administered to asubject (e.g., a mammal, e.g., a human) in an amount sufficient toresult in stimulation of an immune cell in the subject.

In some examples of any of the methods described herein, the immune cellcan be an immature thymocyte, a peripheral blood lymphocyte, a naïve Tcell, a pluripotent Th cell precursor, a lymphoid progenitor cell, aTreg cell, a memory T cell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2cell, a Th1 cell, a Th3 cell, γδ T cell, an αβ T cell, atumor-infiltrating T cell, a CD8⁺ T cell, a CD4⁺ T cell, a naturalkiller T cell, a mast cell, a macrophage, a neutrophil, a dendriticcell, a basophil, an eosinophil, or a natural killer cell, or acombination thereof.

In some examples, the immune cell has previously beengenetically-modified to express a chimeric antigen receptor or arecombinant T-cell receptor. In some examples, the immune cell (e.g.,any of the immune cells described herein) has previously beengenetically-modified to express a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include, after thecontacting step, introducing into the immune cell (e.g., any of theimmune cells described herein) a nucleic acid encoding a chimericantigen-receptor or a recombinant T-cell receptor. Some embodiments ofthese methods can further include, after the contacting step,introducing into the immune cell (e.g., any of the immune cellsdescribed herein) a nucleic acid encoding a co-stimulatory molecule(e.g., CD28).

Some embodiments of these methods can further include administering atherapeutically effective amount of the immune cell to a subject in needthereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosedas having an age-related disease or condition. Non-limiting examples ofage-related diseases or disorders include: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identifiedor diagnosed as having a cancer. Non-limiting examples of cancersinclude: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosedor identified as having an infectious disease. Non-limiting examples ofinfectious disease include infection with human immunodeficiency virus,cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus,smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus,and hepatitis C virus, papillomavirus, or influenza virus.

Detection of the proliferation of an immune cell can be performed usingmethods known in the art, e.g., cytometry (e.g., fluorescence-assistedflow cytometry), microscopy, and immunofluorescence microscopy, e.g., bycomparing the rate of increase in the concentration of the immune cellin a sample not contacted with a single-chain chimeric polypeptide tothe rate of increase in the concentration of the immune cell in asimilar sample contacted with any of the single-chain chimericpolypeptides described herein).

In other examples, the proliferation of an immune cell can be indirectlydetected by detecting an increase in the level of one or more cytokinesor chemokines secreted or regulatory molecules by proliferating immunecells (e.g., one or more of IL-2, IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-7,IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-33,leukotriene B4, CCL5, TNFα, granzymes, perforin, TGFβ, STAT3, RORγT,FOXP3, STATE, and GATA3) (e.g., as compared to the level of the one ormore cytokines and chemokines in a control not contacted with any of thesingle-chain chimeric polypeptides described herein).

In some embodiments, the methods provided herein can result in anincrease (e.g., about 1% to about 800% increase, or any of the subrangesof this range described herein) in the rate of increase in theconcentration of the immune cell in a sample contacted with any of thesingle-chain chimeric polypeptides described herein as compared to therate of increase in a similar control sample not contacted with any ofthe single-chain chimeric polypeptides described herein.

Methods of Inducing Differentiation of an Immune Cell

Also provided herein are method of inducing differentiation of an immunecell (e.g., any of the exemplary immune cells described herein or knownin the art) into a memory or memory-like immune cell that includecontacting an immune cell with an effective amount of any of thesingle-chain chimeric polypeptides described herein or any of thecompositions (e.g., pharmaceutical compositions) described herein. Insome examples, the immune cell is contacted in vitro (e.g., in asuitable liquid culture medium under conditions sufficient to result instimulation of the immune cell).

In some examples, the immune cell has been previously obtained from asubject (e.g., a mammal, e.g., a human). Some embodiments of thesemethods further include obtaining the immune cell from the subject priorto the contacting step.

In some examples, the immune cell is contacted in vivo. In suchembodiments, the single-chain chimeric polypeptide is administered to asubject (e.g., a mammal, e.g., a human) in an amount sufficient toresult in stimulation of an immune cell in the subject.

In some examples of any of the methods described herein, the immune cellcan be an immature thymocyte, a peripheral blood lymphocyte, a naïve Tcell, a pluripotent Th cell precursor, a lymphoid progenitor cell, aTreg cell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell,a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+T cell, a CD4+ T cell, a natural killer T cell, a mast cell, amacrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil,or a natural killer cell, or a combination thereof.

In some examples, the immune cell has previously beengenetically-modified to express a chimeric antigen receptor or arecombinant T-cell receptor. In some examples, the immune cell (e.g.,any of the immune cells described herein) has previously beengenetically-modified to express a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include, after thecontacting step, introducing into the immune cell (e.g., any of theimmune cells described herein) a nucleic acid encoding a chimericantigen-receptor or a recombinant T-cell receptor. Some embodiments ofthese methods can further include, after the contacting step,introducing into the immune cell (e.g., any of the immune cellsdescribed herein) a nucleic acid encoding a co-stimulatory molecule(e.g., CD28).

Some embodiments of these methods can further include administering atherapeutically effective amount of the immune cell to a subject in needthereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosedas having an age-related disease or condition. Non-limiting examples ofage-related diseases or disorders include: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identifiedor diagnosed as having a cancer. Non-limiting examples of cancersinclude: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosedor identified as having an infectious disease. Non-limiting examples ofinfectious disease include infection with human immunodeficiency virus,cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus,smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus,and hepatitis C virus, papillomavirus, or influenza virus.

In some examples, the immune cell is a NK cell, and the detection of amemory NK cell can include, e.g., the detection of the increased levelof one or more of CD25, CD69, CD62L, IL-12, IL-18, IL-33, STAT4, STAT5,Zbtb32, DNAM-1, NKp30, NKp40, NKp46, BIM, Noxa, SOCS1, BNIP3, BNIP3L,IFN-γ, CXCL16, CXCR6, NKG2D, TRAIL, CD49, Ly49D, CD49b, and Ly79H. Adescription of NK memory cells and methods of detecting the same isdescribed in O'Sullivan et al., Immunity 43:634-645, 2015.

In some examples, the immune cell is a T cell, and the detection ofmemory T cells can include, e.g., the detection of the level ofexpression of one or more of CD45RO, CCR7, L-selectin (CD62L), CD44,CD45RA, integrin αβ7, CD43, CD27, CD28, IL-7Rα, CD95, CXCR3, and LFA-1.In some examples, the immune cell is a B cell and the detection ofmemory B cells can include, e.g., the detection of the level ofexpression of CD27. Other types and markers of memory or memory-likeimmune cells are known in the art.

Methods of Treatment

Also provided herein are methods of treating a subject in need thereof(e.g., any of the exemplary subjects described herein or known in theart) that include administering to the subject a therapeuticallyeffective amount of any of the single-chain chimeric polypeptidesdescribed herein or any of the compositions (e.g., pharmaceuticalcompositions) described herein.

In some embodiments of these methods, the subject has been identified ordiagnosed as having a cancer. Non-limiting examples of cancer include:solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma,neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma,B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cellnon-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia(CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML),acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS),cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelialcarcinoma, lung cancer, renal cell carcinoma, gastric and esophagealcancer, pancreatic cancer, prostate cancer, breast cancer, colorectalcancer, ovarian cancer, non-small cell lung carcinoma, squamous cellhead and neck carcinoma, endometrial cancer, cervical cancer, livercancer, and hepatocellular carcinoma. In some embodiments, these methodscan result in a reduction in the number, severity, or frequency of oneor more symptoms of the cancer in the subject (e.g., as compared to thenumber, severity, or frequency of the one or more symptoms of the cancerin the subject prior to treatment). In some embodiments, these methodscan result in a reduction (e.g., about 1% reduction to about 99%reduction, about 1% reduction to about 95% reduction, about 1% reductionto about 90% reduction, about 1% reduction to about 85% reduction, about1% reduction to about 80% reduction, about 1% reduction to about 75%reduction, about 1% reduction to about 70% reduction, about 1% reductionto about 65% reduction, about 1% reduction to about 60% reduction, about1% reduction to about 55% reduction, about 1% reduction to about 50%reduction, about 1% reduction to about 45% reduction, about 1% reductionto about 40% reduction, about 1% reduction to about 35% reduction, about1% reduction to about 30% reduction, about 1% reduction to about 25%reduction, about 1% reduction to about 20% reduction, about 1% reductionto about 15% reduction, about 1% reduction to about 10% reduction, about1% reduction to about 5% reduction, about 5% reduction to about 99%reduction, about 5% reduction to about 95% reduction, about 5% reductionto about 90% reduction, about 5% reduction to about 85% reduction, about5% reduction to about 80% reduction, about 5% reduction to about 75%reduction, about 5% reduction to about 70% reduction, about 5% reductionto about 65% reduction, about 5% reduction to about 60% reduction, about5% reduction to about 55% reduction, about 5% reduction to about 50%reduction, about 5% reduction to about 45% reduction, about 5% reductionto about 40% reduction, about 5% reduction to about 35% reduction, about5% reduction to about 30% reduction, about 5% reduction to about 25%reduction, about 5% reduction to about 20% reduction, about 5% reductionto about 15% reduction, about 5% reduction to about 10% reduction, about10% reduction to about 99% reduction, about 10% reduction to about 95%reduction, about 10% reduction to about 90% reduction, about 10%reduction to about 85% reduction, about 10% reduction to about 80%reduction, about 10% reduction to about 75% reduction, about 10%reduction to about 70% reduction, about 10% reduction to about 65%reduction, about 10% reduction to about 60% reduction, about 10%reduction to about 55% reduction, about 10% reduction to about 50%reduction, about 10% reduction to about 45% reduction, about 10%reduction to about 40% reduction, about 10% reduction to about 35%reduction, about 10% reduction to about 30% reduction, about 10%reduction to about 25% reduction, about 10% reduction to about 20%reduction, about 10% reduction to about 15% reduction, about 15%reduction to about 99% reduction, about 15% reduction to about 95%reduction, about 15% reduction to about 90% reduction, about 15%reduction to about 85% reduction, about 15% reduction to about 80%reduction, about 15% reduction to about 75% reduction, about 15%reduction to about 70% reduction, about 15% reduction to about 65%reduction, about 15% reduction to about 60% reduction, about 15%reduction to about 55% reduction, about 15% reduction to about 50%reduction, about 15% reduction to about 45% reduction, about 15%reduction to about 40% reduction, about 15% reduction to about 35%reduction, about 15% reduction to about 30% reduction, about 15%reduction to about 25% reduction, about 15% reduction to about 20%reduction, about 20% reduction to about 99% reduction, about 20%reduction to about 95% reduction, about 20% reduction to about 90%reduction, about 20% reduction to about 85% reduction, about 20%reduction to about 80% reduction, about 20% reduction to about 75%reduction, about 20% reduction to about 70% reduction, about 20%reduction to about 65% reduction, about 20% reduction to about 60%reduction, about 20% reduction to about 55% reduction, about 20%reduction to about 50% reduction, about 20% reduction to about 45%reduction, about 20% reduction to about 40% reduction, about 20%reduction to about 35% reduction, about 20% reduction to about 30%reduction, about 20% reduction to about 25% reduction, about 25%reduction to about 99% reduction, about 25% reduction to about 95%reduction, about 25% reduction to about 90% reduction, about 25%reduction to about 85% reduction, about 25% reduction to about 80%reduction, about 25% reduction to about 75% reduction, about 25%reduction to about 70% reduction, about 25% reduction to about 65%reduction, about 25% reduction to about 60% reduction, about 25%reduction to about 55% reduction, about 25% reduction to about 50%reduction, about 25% reduction to about 45% reduction, about 25%reduction to about 40% reduction, about 25% reduction to about 35%reduction, about 25% reduction to about 30% reduction, about 30%reduction to about 99% reduction, about 30% reduction to about 95%reduction, about 30% reduction to about 90% reduction, about 30%reduction to about 85% reduction, about 30% reduction to about 80%reduction, about 30% reduction to about 75% reduction, about 30%reduction to about 70% reduction, about 30% reduction to about 65%reduction, about 30% reduction to about 60% reduction, about 30%reduction to about 55% reduction, about 30% reduction to about 50%reduction, about 30% reduction to about 45% reduction, about 30%reduction to about 40% reduction, about 30% reduction to about 35%reduction, about 35% reduction to about 99% reduction, about 35%reduction to about 95% reduction, about 35% reduction to about 90%reduction, about 35% reduction to about 85% reduction, about 35%reduction to about 80% reduction, about 35% reduction to about 75%reduction, about 35% reduction to about 70% reduction, about 35%reduction to about 65% reduction, about 35% reduction to about 60%reduction, about 35% reduction to about 55% reduction, about 35%reduction to about 50% reduction, about 35% reduction to about 45%reduction, about 35% reduction to about 40% reduction, about 40%reduction to about 99% reduction, about 40% reduction to about 95%reduction, about 40% reduction to about 90% reduction, about 40%reduction to about 85% reduction, about 40% reduction to about 80%reduction, about 40% reduction to about 75% reduction, about 40%reduction to about 70% reduction, about 40% reduction to about 65%reduction, about 40% reduction to about 60% reduction, about 40%reduction to about 55% reduction, about 40% reduction to about 50%reduction, about 40% reduction to about 45% reduction, about 45%reduction to about 99% reduction, about 45% reduction to about 95%reduction, about 45% reduction to about 90% reduction, about 45%reduction to about 85% reduction, about 45% reduction to about 80%reduction, about 45% reduction to about 75% reduction, about 45%reduction to about 70% reduction, about 45% reduction to about 65%reduction, about 45% reduction to about 60% reduction, about 45%reduction to about 55% reduction, about 45% reduction to about 50%reduction, about 50% reduction to about 99% reduction, about 50%reduction to about 95% reduction, about 50% reduction to about 90%reduction, about 50% reduction to about 85% reduction, about 50%reduction to about 80% reduction, about 50% reduction to about 75%reduction, about 50% reduction to about 70% reduction, about 50%reduction to about 65% reduction, about 50% reduction to about 60%reduction, about 50% reduction to about 55% reduction, about 55%reduction to about 99% reduction, about 55% reduction to about 95%reduction, about 55% reduction to about 90% reduction, about 55%reduction to about 85% reduction, about 55% reduction to about 80%reduction, about 55% reduction to about 75% reduction, about 55%reduction to about 70% reduction, about 55% reduction to about 65%reduction, about 55% reduction to about 60% reduction, about 60%reduction to about 99% reduction, about 60% reduction to about 95%reduction, about 60% reduction to about 90% reduction, about 60%reduction to about 85% reduction, about 60% reduction to about 80%reduction, about 60% reduction to about 75% reduction, about 60%reduction to about 70% reduction, about 60% reduction to about 65%reduction, about 65% reduction to about 99% reduction, about 65%reduction to about 95% reduction, about 65% reduction to about 90%reduction, about 65% reduction to about 85% reduction, about 65%reduction to about 80% reduction, about 65% reduction to about 75%reduction, about 65% reduction to about 70% reduction, about 70%reduction to about 99% reduction, about 70% reduction to about 95%reduction, about 70% reduction to about 90% reduction, about 70%reduction to about 85% reduction, about 70% reduction to about 80%reduction, about 70% reduction to about 75% reduction, about 75%reduction to about 99% reduction, about 75% reduction to about 95%reduction, about 75% reduction to about 90% reduction, about 75%reduction to about 85% reduction, about 75% reduction to about 80%reduction, about 80% reduction to about 99% reduction, about 80%reduction to about 95% reduction, about 80% reduction to about 90%reduction, about 80% reduction to about 85% reduction, about 85%reduction to about 99% reduction, about 85% reduction to about 95%reduction, about 85% reduction to about 90% reduction, about 90%reduction to about 99% reduction, about 90% reduction to about 95%reduction, or about 95% reduction to about 99% reduction) in the volumeof one or more solid tumors in the subject (e.g., as compared to thevolume of the one or more solid tumors prior to treatment or at thestart of treatment). In some embodiments, the these methods can reduce(e.g., about 1% reduction to about 99% reduction, or any of thesubranges of this range described herein) the risk of developing ametastasis or developing one or more additional metastasis in a subject(e.g., as compared to the risk of developing a metastasis or developingone or more additional metastasis in a subject prior to treatment or ina similar subject or a population of subjects administered a differenttreatment).

In some examples of these methods, the subject has been identified ordiagnosed as having an aging-related disease or condition. Non-limitingexamples of aging-related diseases and conditions include Alzheimer'sdisease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction. In some examples, thesemethods can result in a reduction in the number, severity, or frequencyof one or more symptoms of the aging-related disease or condition in thesubject (e.g., as compared to the number, severity, or frequency of theone or more symptoms of the aging-related disease or condition in thesubject prior to treatment). In some examples, the methods can result ina decrease (e.g., about 1% decrease to about 99% decrease, an about 1%decrease to about 95% decrease, about 1% decrease to about 90% decrease,about 1% decrease to about 85% decrease, about 1% decrease to about 80%decrease, about 1% decrease to about 75% decrease, about 1% to about 70%decrease, about 1% decrease to about 65% decrease, about 1% decrease toabout 60% decrease, about 1% decrease to about 55% decrease, about 1%decrease to about 50% decrease, about 1% decrease to about 45% decrease,about 1% decrease to about 40% decrease, about 1% decrease to about 35%decrease, about 1% decrease to about 30% decrease, about 1% decrease toabout 25% decrease, about 1% decrease to about 20% decrease, about 1%decrease to about 15% decrease, about 1% decrease to about 10% decrease,about 1% decrease to about 5% decrease, about 5% decrease to about 99%decrease, an about 5% decrease to about 95% decrease, about 5% decreaseto about 90% decrease, about 5% decrease to about 85% decrease, about 5%decrease to about 80% decrease, about 5% decrease to about 75% decrease,about 5% to about 70% decrease, about 5% decrease to about 65% decrease,about 5% decrease to about 60% decrease, about 5% decrease to about 55%decrease, about 5% decrease to about 50% decrease, about 5% decrease toabout 45% decrease, about 5% decrease to about 40% decrease, about 5%decrease to about 35% decrease, about 5% decrease to about 30% decrease,about 5% decrease to about 25% decrease, about 5% decrease to about 20%decrease, about 5% decrease to about 15% decrease, about 5% decrease toabout 10% decrease, about 10% decrease to about 99% decrease, an about10% decrease to about 95% decrease, about 10% decrease to about 90%decrease, about 10% decrease to about 85% decrease, about 10% decreaseto about 80% decrease, about 10% decrease to about 75% decrease, about10% to about 70% decrease, about 10% decrease to about 65% decrease,about 10% decrease to about 60% decrease, about 10% decrease to about55% decrease, about 10% decrease to about 50% decrease, about 10%decrease to about 45% decrease, about 10% decrease to about 40%decrease, about 10% decrease to about 35% decrease, about 10% decreaseto about 30% decrease, about 10% decrease to about 25% decrease, about10% decrease to about 20% decrease, about 10% decrease to about 15%decrease, about 15% decrease to about 99% decrease, an about 15%decrease to about 95% decrease, about 15% decrease to about 90%decrease, about 15% decrease to about 85% decrease, about 15% decreaseto about 80% decrease, about 15% decrease to about 75% decrease, about15% to about 70% decrease, about 15% decrease to about 65% decrease,about 15% decrease to about 60% decrease, about 15% decrease to about55% decrease, about 15% decrease to about 50% decrease, about 15%decrease to about 45% decrease, about 15% decrease to about 40%decrease, about 15% decrease to about 35% decrease, about 15% decreaseto about 30% decrease, about 15% decrease to about 25% decrease, about15% decrease to about 20% decrease, about 20% decrease to about 99%decrease, an about 20% decrease to about 95% decrease, about 20%decrease to about 90% decrease, about 20% decrease to about 85%decrease, about 20% decrease to about 80% decrease, about 20% decreaseto about 75% decrease, about 20% to about 70% decrease, about 20%decrease to about 65% decrease, about 20% decrease to about 60%decrease, about 20% decrease to about 55% decrease, about 20% decreaseto about 50% decrease, about 20% decrease to about 45% decrease, about20% decrease to about 40% decrease, about 20% decrease to about 35%decrease, about 20% decrease to about 30% decrease, about 20% decreaseto about 25% decrease, about 25% decrease to about 99% decrease, anabout 25% decrease to about 95% decrease, about 25% decrease to about90% decrease, about 25% decrease to about 85% decrease, about 25%decrease to about 80% decrease, about 25% decrease to about 75%decrease, about 25% to about 70% decrease, about 25% decrease to about65% decrease, about 25% decrease to about 60% decrease, about 25%decrease to about 55% decrease, about 25% decrease to about 50%decrease, about 25% decrease to about 45% decrease, about 25% decreaseto about 40% decrease, about 25% decrease to about 35% decrease, about25% decrease to about 30% decrease, about 30% decrease to about 99%decrease, an about 30% decrease to about 95% decrease, about 30%decrease to about 90% decrease, about 30% decrease to about 85%decrease, about 30% decrease to about 80% decrease, about 30% decreaseto about 75% decrease, about 30% to about 70% decrease, about 30%decrease to about 65% decrease, about 30% decrease to about 60%decrease, about 30% decrease to about 55% decrease, about 30% decreaseto about 50% decrease, about 30% decrease to about 45% decrease, about30% decrease to about 40% decrease, about 30% decrease to about 35%decrease, about 35% decrease to about 99% decrease, an about 35%decrease to about 95% decrease, about 35% decrease to about 90%decrease, about 35% decrease to about 85% decrease, about 35% decreaseto about 80% decrease, about 35% decrease to about 75% decrease, about35% to about 70% decrease, about 35% decrease to about 65% decrease,about 35% decrease to about 60% decrease, about 35% decrease to about55% decrease, about 35% decrease to about 50% decrease, about 35%decrease to about 45% decrease, about 35% decrease to about 40%decrease, about 40% decrease to about 99% decrease, an about 40%decrease to about 95% decrease, about 40% decrease to about 90%decrease, about 40% decrease to about 85% decrease, about 40% decreaseto about 80% decrease, about 40% decrease to about 75% decrease, about40% to about 70% decrease, about 40% decrease to about 65% decrease,about 40% decrease to about 60% decrease, about 40% decrease to about55% decrease, about 40% decrease to about 50% decrease, about 40%decrease to about 45% decrease, about 45% decrease to about 99%decrease, an about 45% decrease to about 95% decrease, about 45%decrease to about 90% decrease, about 45% decrease to about 85%decrease, about 45% decrease to about 80% decrease, about 45% decreaseto about 75% decrease, about 45% to about 70% decrease, about 45%decrease to about 65% decrease, about 45% decrease to about 60%decrease, about 45% decrease to about 55% decrease, about 45% decreaseto about 50% decrease, about 50% decrease to about 99% decrease, anabout 50% decrease to about 95% decrease, about 50% decrease to about90% decrease, about 50% decrease to about 85% decrease, about 50%decrease to about 80% decrease, about 50% decrease to about 75%decrease, about 50% to about 70% decrease, about 50% decrease to about65% decrease, about 50% decrease to about 60% decrease, about 50%decrease to about 55% decrease, about 55% decrease to about 99%decrease, an about 55% decrease to about 95% decrease, about 55%decrease to about 90% decrease, about 55% decrease to about 85%decrease, about 55% decrease to about 80% decrease, about 55% decreaseto about 75% decrease, about 55% to about 70% decrease, about 55%decrease to about 65% decrease, about 55% decrease to about 60%decrease, about 60% decrease to about 99% decrease, an about 60%decrease to about 95% decrease, about 60% decrease to about 90%decrease, about 60% decrease to about 85% decrease, about 60% decreaseto about 80% decrease, about 60% decrease to about 75% decrease, about60% to about 70% decrease, about 60% decrease to about 65% decrease,about 65% decrease to about 99% decrease, an about 65% decrease to about95% decrease, about 65% decrease to about 90% decrease, about 65%decrease to about 85% decrease, about 65% decrease to about 80%decrease, about 65% decrease to about 75% decrease, about 65% to about70% decrease, about 70% decrease to about 99% decrease, an about 70%decrease to about 95% decrease, about 70% decrease to about 90%decrease, about 70% decrease to about 85% decrease, about 70% decreaseto about 80% decrease, about 70% decrease to about 75% decrease, about75% decrease to about 99% decrease, an about 75% decrease to about 95%decrease, about 75% decrease to about 90% decrease, about 75% decreaseto about 85% decrease, about 75% decrease to about 80% decrease, about80% decrease to about 99% decrease, an about 80% decrease to about 95%decrease, about 80% decrease to about 90% decrease, about 80% decreaseto about 85% decrease, about 85% decrease to about 99% decrease, anabout 85% decrease to about 95% decrease, about 85% decrease to about90% decrease, about 90% decrease to about 99% decrease, an about 90%decrease to about 95% decrease, or about 95% decrease to about 99%decrease) in the number of senescent cells in the subject (e.g., adecrease in the number of senescent cells in one or more specifictissues involved and/or implicated in the aging-related disease ordisorder in the subject), e.g., as compared to the number of senescentcells in the subject prior to treatment.

In some examples of these methods, the subject has been diagnosed oridentified as having an infectious disease. Non-limiting examples ofinfectious disease include infection with human immunodeficiency virus,cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus,smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus,and hepatitis C virus, papillomavirus, and influenza virus. In someembodiments, these methods can result in a decrease in the infectioustiter (e.g., viral titer) in a subject (e.g., as compared to theinfectious titer in the subject prior to treatment). In someembodiments, these methods can result in a reduction in the number,severity, or frequency of one or more symptoms of the infectious disease(e.g., viral infection) in the subject (e.g., as compared to the number,severity, or frequency of the one or more symptoms of the infectiousdisease in the subject prior to treatment).

The term “subject” refers to any mammal. In some embodiments, thesubject or “subject in need of treatment” may be a canine (e.g., a dog),feline (e.g., a cat), equine (e.g., a horse), ovine, bovine, porcine,caprine, primate, e.g., a simian (e.g., a monkey (e.g., marmoset,baboon), or an ape (e.g., a gorilla, chimpanzee, orangutan, or gibbon)or a human; or rodent (e.g., a mouse, a guinea pig, a hamster, or arat). In some embodiments, the subject or “subject in need of treatment”may be a non-human mammal, especially mammals that are conventionallyused as models for demonstrating therapeutic efficacy in humans (e.g.,murine, lapine, porcine, canine or primate animals) may be employed.

Methods of Killing a Cancer Cell, an Infected Cell, or a Senescent Cell

Also provided herein are methods of killing a cancer cell (e.g., any ofthe exemplary types of cancer described herein or known in the art), aninfected cell (e.g., a cell infected with any of the exemplary virusesdescribed herein or known in the art), or a senescent cell (e.g., asenescent cancer cell, a senescent fibroblast, or a senescentendothelial cell) in a subject in need thereof (e.g., any of theexemplary subjects described herein or known in the art) that includeadministering to the subject a therapeutically effective amount of anyof the single-chain chimeric polypeptides described herein or any of thecompositions (e.g., pharmaceutical compositions) described herein.

In some embodiments of these methods, the subject has been identified ordiagnosed as having a cancer. Non-limiting examples of cancer include:solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma,neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma,B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cellnon-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia(CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML),acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS),cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelialcarcinoma, lung cancer, renal cell carcinoma, gastric and esophagealcancer, pancreatic cancer, prostate cancer, breast cancer, colorectalcancer, ovarian cancer, non-small cell lung carcinoma, squamous cellhead and neck carcinoma, endometrial cancer, cervical cancer, livercancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been identified ordiagnosed as having an aging-related disease or condition. Non-limitingexamples of aging-related diseases and conditions include Alzheimer'sdisease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been diagnosed oridentified as having an infectious disease. Non-limiting examples of aninfectious disease include infection with human immunodeficiency virus,cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus,smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus,and hepatitis C virus, papillomavirus, and influenza virus.

Senescent Cells

Senescence is a form of irreversible growth arrest accompanied byphenotypic changes, resistance to apoptosis and activation ofdamage-sensing signaling pathways. Cellular senescence was firstdescribed in cultured human fibroblast cells that lost their ability toproliferate, reaching permanent arrest after about 50 populationdoublings (referred to as the Hayflick limit). Senescence is considereda stress response that can be induced by a wide range of intrinsic andextrinsic insults, including oxidative and genotoxic stress, DNA damage,telomere attrition, oncogenic activation, mitochondrial dysfunction, orchemotherapeutic agents.

Senescent cells remain metabolically active and can influence the tissuehemostasis, disease and aging through their secretory phenotype.Senescence is considered as a physiologic process and is important inpromoting wound healing, tissue homeostasis, regeneration, and fibrosisregulation. For instance, transient induction of senescent cells isobserved during would healing and contributes to wound resolution.Perhaps one of the most important roles of senescence is its role intumor suppression. However, the accumulation of senescent cells alsodrives aging- and aging-related diseases and conditions. The senescentphenotype also can trigger chronic inflammatory responses andconsequently augment chronic inflammatory conditions to promote tumorgrowth. The connection between senescence and aging was initially basedon observations that senescent cells accumulate in aged tissue. The useof transgenic models has enabled the detection of senescent cellssystematically in many age-related pathologies. Strategies toselectively eliminate senescent cells has demonstrated that senescentcells can indeed play a causal role in aging and related pathologies.

Senescent cells display important and unique properties which includechanges in morphology, chromatin organization, gene expression, andmetabolism. There are several biochemical and functional propertiesassociated with cellular senescence, such as (i) increased expression ofp16 and p21, inhibitors of cyclin-dependent kinases, (ii) presence ofsenescence-associated β-galactosidase, a marker of lysosomal activity,(iii) appearance of senescence-associated heterochromatin foci anddownregulation of lamin B1 levels, (iv) resistance to apoptosis causedby an increased expression of anti-apoptotic BCL-family protein, and (v)upregulation of CD26 (DPP4), CD36 (Scavenger receptor), forkhead box 4(FOXO4), and secretory carrier membrane protein 4 (SCAMP4). Senescentcells also express an inflammatory signature, the so-calledsenescence-associated secretory phenotype (SASP). Through SASP, thesenescent cells produce a wide range of inflammatory cytokines (IL-6,IL-8), growth factors (TGF-β), chemokines (CCL-2), and matrixmetalloproteinases (MMP-3, MMP-9) that operate in a cell-autonomousmanner to reinforce senescence (autocrine effects) and communicate withand modify the microenvironment (paracrine effects). SASP factors cancontribute to tumor suppression by triggering senescence surveillance,an immune-mediated clearance of senescent cells. However, chronicinflammation is also a known driver of tumorigenesis, and accumulatingevidence indicates that chronic SASP can also boost cancer andaging-related diseases.

The secretion profile of senescent cells is context dependent. Forinstance, the mitochondrial dysfunction-associated senescence (MiDAS),induced by different mitochondrial dysfunction in human fibroblasts, ledto the appearance of a SASP that was deficient in IL-1-dependentinflammatory factors. A decrease in the NAD+/NADH ratio activated AMPKsignaling which induced MiDAS through the activation of p53. As aresult, p53 inhibited NF-κB signaling which is a crucial inducer ofpro-inflammatory SASP. In contrast, the cellular senescence caused bypersistent DNA damage in human cells induced an inflammatory SASP, whichwas dependent on the activation of ataxia-telangiectasia mutated (ATM)kinase but not on that of p53. In particular, the expression andsecretion levels of IL-6 and IL-8 were increased. It was alsodemonstrated that cellular senescence caused by the ectopic expressionp16INK4a and p21CIP1 induced the senescent phenotype in humanfibroblasts without an inflammatory SASP indicating that the growtharrest itself did not stimulate SASP.

One of the most defining characteristics of senescence is stable growtharrest. This is achieved by two important pathways, the p16/Rb and thep53/p21, both of which are central in tumor suppression. DNA damageresults in: (1) high deposition of γH2Ax (histone coding gene) and 53BP1(involved in DNA damage response) in chromatin: this leads to activationof a kinase cascade eventually resulting in p53 activation, and (2)activation of p16INK4a and ARF (both encoded by CDKN2A) and P15INK4b(encoded by CDKN2B): p53 induces transcription of cyclin-dependentkinase inhibitor (p21) and along with both p16INK4a and p15INK4b blockgenes for cell cycle progression (CDK4 and CDK6). This eventually leadsto hypophosphorylation of Retinoblastoma protein (Rb) and cell cyclearrest at the G1 phase.

Selectively killing senescent cells has been shown to significantlyimprove the health span of mice in the context of normal aging andameliorates the consequences of age-related disease or cancer therapy(Ovadya, J Clin Invest. 128(4):1247-1254, 2018). In nature, thesenescent cells are normally removed by the innate immune cells.Induction of senescence not only prevents the potential proliferationand transformation of damaged/altered cells, but also favors tissuerepair through the production of SASP factors that function aschemoattractants mainly for Natural Killer (NK) cells (such as IL-15 andCCL2) and macrophages (such as CFS-1 and CCL2). These innate immunecells mediate the immunosurveillance mechanism for eliminating stressedcells. Senescent cells usually up-regulate the NK-cell activatingreceptor NKG2D and DNAM1 ligands, which belong to a family ofstress-inducible ligands: an important component of the frontline immunedefense against infectious diseases and malignancies. Upon receptoractivation, NK cells can then specifically induce the death of senescentcells through their cytolytic machinery. A role for NK cells in theimmune surveillance of senescent cells has been pointed out in liverfibrosis (Sagiv, Oncogene 32(15): 1971-1977, 2013), hepatocellularcarcinoma (Iannello, J Exp Med 210(10): 2057-2069, 2013), multiplemyeloma (Soriani, Blood 113(15): 3503-3511, 2009), and glioma cellsstressed by dysfunction of the mevalonate pathway (Ciaglia, Int J Cancer142(1): 176-190, 2018). Endometrial cells undergo acute cellularsenescence and do not differentiate into decidual cells. Thedifferentiated decidual cells secrete IL-15 and thereby recruit uterineNK cells to target and eliminate the undifferentiated senescent cellsthus helping to re-model and rejuvenate the endometrium (Brighton, Elife6: e31274, 2017). With a similar mechanism, during liver fibrosis,p53-expressing senescent liver satellite cells skewed the polarizationof resident Kupfer macrophages and freshly infiltrated macrophagestoward the pro-inflammatory M1 phenotype, which display senolyticactivity. F4/80+ macrophages have been shown to play a key role in theclearance of mouse uterine senescent cells to maintain postpartumuterine function.

Senescent cells recruit NK cells by mainly upregulating ligands to NKG2D(expressed on NK cells), chemokines, and other SASP factors. In vivomodels of liver fibrosis have shown effective clearance of senescentcells by activated NK cells (Krizhanovsky, Cell 134(4): 657-667, 2008).Studies have described various models to study senescence includingliver fibrosis (Krizhanovsky, Cell 134(4): 657-667, 2008),osteoarthritis (Xu, J Gerontol A Biol Sci Med Sci 72(6): 780-785, 2017),and Parkinson's disease (Chinta, Cell Rep 22(4): 930-940, 2018). Animalmodels for studying senescent cells are described in: Krizhanovsky, Cell134(4): 657-667, 2008; Baker, Nature 479(7372): 232-236, 2011; Farr, NatMed 23(9): 1072-1079, 2017; Bourgeois, FEBS Lett 592(12): 2083-2097,2018; Xu, Nat Med 24(8): 1246-1256, 2018).

Additional Therapeutic Agents

Some embodiments of any of the methods described herein can furtherinclude administering to a subject (e.g., any of the subjects describedherein) a therapeutically effective amount of one or more additionaltherapeutic agents. The one or more additional therapeutic agents can beadministered to the subject at substantially the same time as asingle-chain chimeric polypeptide (e.g., any of the single-chainchimeric polypeptides described herein) or an immune cell (e.g.,administered as a single formulation or two or more formulations to thesubject). In some embodiments, one or more additional therapeutic agentscan be administered to the subject prior to administration of asingle-chain chimeric polypeptide (e.g., any of the single-chainchimeric polypeptides described herein) or an immune cell. In someembodiments, one or more additional therapeutic agents can beadministered to the subject after administration of a single-chainchimeric polypeptide (e.g., any of the single-chain chimericpolypeptides described herein) or an immune cell to the subject.

Non-limiting examples of additional therapeutic agents include:anti-cancer drugs, activating receptor agonists, immune checkpointinhibitors, agents for blocking HLA-specific inhibitory receptors,Glucogen Synthase Kinase (GSK) 3 inhibitors, and antibodies.

Non-limiting examples of anticancer drugs include antimetabolic drugs(e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine,cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide,methotrexate, 6-thioguanine, cladribine, nelarabine, pentostatin, orpemetrexed), plant alkaloids (e.g., vinblastine, vincristine, vindesine,camptothecin, 9-methoxycamptothecin, coronaridine, taxol, naucleaorals,diprenylated indole alkaloid, montamine, schischkiniin, protoberberine,berberine, sanguinarine, chelerythrine, chelidonine, liriodenine,clivorine, β-carboline, antofine, tylophorine, cryptolepine,neocryptolepine, corynoline, sampangine, carbazole, crinamine,montanine, ellipticine, paclitaxel, docetaxel, etoposide, tenisopide,irinotecan, topotecan, or acridone alkaloids), proteasome inhibitors(e.g., lactacystin, disulfiram, epigallocatechin-3-gallate, marizomib(salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770),epoxomicin, MG132, beta-hydroxy beta-methylbutyrate, bortezomib,carfilzomib, or ixazomib), antitumor antibiotics (e.g., doxorubicin,daunorubicin, epirubicin, mitoxantrone, idarubicin, actinomycin,plicamycin, mitomycin, or bleomycin), histone deacetylase inhibitors(e.g., vorinostat, panobinostat, belinostat, givinostat, abexinostat,depsipeptide, entinostat, phenyl butyrate, valproic acid, trichostatinA, dacinostat, mocetinostat, pracinostat, nicotinamide, cambinol,tenovin 1, tenovin 6, sirtinol, ricolinostat, tefinostat, kevetrin,quisinostat, resminostat, tacedinaline, chidamide, or selisistat),tyrosine kinase inhibitors (e.g., axitinib, dasatinib, encorafinib,erlotinib, imatinib, nilotinib, pazopanib, and sunitinib), andchemotherapeutic agents (e.g., all-trans retinoic acid, azacitidine,azathioprine, doxifluridine, epothilone, hydroxyurea, imatinib,teniposide, tioguanine, valrubicin, vemurafenib, and lenalidomide).Additional examples of chemotherapeutic agents include alkylatingagents, e.g., mechlorethamine, cyclophosphamide, chlorambucil,melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan,altretamine, procarbazine, dacarbazine, temozolomide, carmustine,lumustine, streptozocin, carboplatin, cisplatin, and oxaliplatin.

Non-limiting examples of activating receptor agonists include anyagonists for activating receptors which activate and enhance thecytotoxicity of NK cells, including anti-CD16 antibodies (e.g.,anti-CD16/CD30 bispecific monoclonal antibody (BiMAb)) and Fc-basedfusion proteins. Non-limiting examples of checkpoint inhibitors includeanti-PD-1 antibodies (e.g., MEDI0680), anti-PD-L1 antibodies (e.g.,BCD-135, BGB-A333, CBT-502, CK-301, CS1001, FAZ053, KN035, MDX-1105,MSB2311, SHR-1316, anti-PD-L1/CTLA-4 bispecific antibody KN046,anti-PD-L1/TGFβRII fusion protein M7824, anti-PD-L1/TIM-3 bispecificantibody LY3415244, atezolizumab, or avelumab), anti-TIM3 antibodies(e.g., TSR-022, Sym023, or MBG453) and anti-CTLA-4 antibodies (e.g.,AGEN1884, MK-1308, or an anti-CTLA-4/OX40 bispecific antibodyATOR-1015). Non-limiting examples of agents for blocking HLA-specificinhibitory receptors include monalizumab (e.g., an anti-HLA-E NKG2Ainhibitory receptor monoclonal antibody). Non-limiting examples of GSK3inhibitor include tideglusib or CHIR99021. Non-limiting examples ofantibodies that can be used as additional therapeutic agents includeanti-CD26 antibodies (e.g., YS110), anti-CD36 antibodies, and any otherantibody or antibody construct that can bind to and activate an Fcreceptor (e.g., CD16) on a NK cell. In some embodiments, an additionaltherapeutic agent can be insulin or metformin.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1. Production of an Exemplary Single-Chain Chimeric Polypeptides

An exemplary single-chain chimeric polypeptide including a firsttarget-binding domain that is an anti-CD3 scFv, a soluble human tissuefactor domain, and a second target-binding domain that is an anti-CD28scFv was generated (αCD3scFv/TF/αCD28scFv) (FIG. 1 ). The nucleic acidand amino acid sequences of this single-chain chimeric polypeptide areshown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide(αCD3scFv/TF/αCD28scFv) (SEQ ID NO: 4) (Signal peptide)ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCT ATTCC(αCD3 light chain variable region)CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCT CGAAATCAATCGT(Linker) GGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGC(αCD3 heavy chain variable region)CAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGC(Human tissue factor 219 form)TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (αCD28 light chain variable region)GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGC (Linker)GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCC(αCD28 light chain variable region)GACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGAC AAAGAGGExemplary Single-Chain Chimeric Polypeptide (αCD3scFv/TF/αCD28scFv)(SEQ ID NO: 3) (Signal peptide) MKWVTFISLLFLFSSAYS(αCD3 light chain variable region)QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI NR (Linker)GGGGSGGGGSGGGGS (αCD3 heavy chain variable region)QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD HYCLDYWGQGTTLTVSS(Human tissue factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE(αCD28 light chain variable region)VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNY WGRGTTLTVSS(Linker) GGGGSGGGGSGGGGS (αCD28 heavy chain variable region)DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR

A second exemplary single-chain chimeric polypeptide including a firsttarget-binding domain that is an anti-CD28 scFv, a soluble human tissuefactor domain, and a second target-binding domain that is an anti-CD3scFv was generated (αCD28scFv/TF/αCD3scFv) (FIG. 2 ). The nucleic acidand amino acid sequences of this single-chain chimeric polypeptide areshown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide(αCD28scFv/TF/αCD3scFv) (SEQ ID NO: 8) (Signal peptide)ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTTATTTAGCAGCGCCT ACAGC(αCD28 light chain variable region)GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGCCGGGGAACTACTTTAACAGTGAGCTCC (Linker)GGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGC(αCD28 heavy chain variable region)GACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAG ACCAAGCGG(Human tissue factor 219 form)AGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACCGACGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCCTACCCCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGAGAGTTTCGTGAG (αCD3 light chain variable region)CAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTC GAGATTAATCGT(Linker) GGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGGAGGTAGC(αCD3 heavy chain variable region)CAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCCExemplary Single-Chain Chimeric Polypeptide (αCD28scFv/TF/CD3scFv)(SEQ ID NO: 7) (Signal peptide) MKWVTFISLLFLFSSAYS(αCD28 light chain variable region)VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNY WGRGTTLTVSS(Linker) GGGGSGGGGSGGGGS (αCD28 heavy chain variable region)DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR(Human tissue factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE(αCD3 light chain variable region)QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI NR (Linker)GGGGSGGGGSGGGGS (αCD3 heavy chain variable region)QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD HYCLDYWGQGTTLTVSS

The nucleic acid encoding αCD3scFv/TF/αCD28scFv was cloned into amodified retrovirus expression vectors as described previously (Hugheset al., Hum Gene Ther 16:457-72, 2005). The expression vector encodingαCD3scFv/TF/αCD28scFv was transfected into CHO-K1 cells. Expression ofthe expression vector in CHO-K1 cells allowed for secretion of thesoluble αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide(referred to as 3t28), which can be purified by anti-TF antibodyaffinity and other chromatography methods.

An anti-tissue factor antibody affinity column was used to purify theαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide. The anti-tissuefactor antibody affinity column was connected to a GE Healthcare AKTAAvant system. A flow rate of 4 mL/min was used for all steps except theelution step, which was 2 mL/min.

Cell culture harvest including αCD3scFv/TF/αCD28scFv single-chainchimeric polypeptide was adjusted to pH 7.4 with 1M Tris base and loadedonto the anti-TF antibody affinity column (described above) which wasequilibrated with 5 column volumes of PBS. After sample loading, thecolumn was washed with 5 column volumes PBS, followed by elution with 6column volumes 0.1 M acetic acid, pH 2.9. An A280 elution peak wascollected and then neutralized to pH 7.5-8.0 by adding 1 M Tris base.The neutralized sample was then buffer exchanged into PBS using Amiconcentrifugal filters with a 30 kDa molecular weight cutoff. The data inFIG. 2 show that the anti-tissue factor antibody affinity column canbind the αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide, whichcontains a human soluble tissue factor domain. The buffer-exchangedprotein sample was stored at 2-8° C. for further biochemical analysisand biological activity testing.

After each elution, the anti-tissue factor antibody affinity column wasstripped using 6 column volumes of 0.1 M glycine, pH 2.5. The column wasthen neutralized using 10 column volumes of PBS, 0.05% NaN₃, and storedat 2-8° C.

Analytical size exclusion chromatography (SEC) was performed on theαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide using a Superdex200 Increase 10/300 GL gel filtration column (from GE Healthcare)connected to an AKTA Avant system (from GE Healthcare). The column wasequilibrated with 2 column volumes of PBS. A flow rate of 0.8 mL/min wasused. Two hundred μL of αCD3scFv/TF/αCD28scFv single-chain chimericpolypeptide (1 mg/mL) was injected onto the column using a capillaryloop. After injection of the single-chain chimeric polypeptide, 1.25column volumes of PBS were flowed into the column. The SEC chromatographis shown in FIG. 3 . The data show that there are 3 protein peaks,likely representing a monomer and dimer or other different forms of theαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide.

To determine the purity and protein molecular weight of theαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide, the purifiedαCD3scFv/TF/αCD28scFv protein sample from anti-tissue factor antibodyaffinity column was analyzed by standard sodium dodecyl sulfatepolyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis(SDS-PAGE) method under reduced conditions. The gel was stained withInstantBlue for about 30 minutes and destained overnight with purifiedwater. FIG. 4 shows the SDS gel of the αCD3scFv/TF/αCD28scFvsingle-chain chimeric polypeptide purified using an anti-tissue factorantibody affinity column. The results show that the purifiedαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide has the expectedmolecular weight (72 kDa) in reduced SDS gel.

Example 2. Functional Characterization of αCD3scFv/TF/αCD28scFvSingle-Chain Chimeric Polypeptide

ELISA-based methods confirmed the formation of the αCD3scFv/TF/αCD28scFvsingle-chain chimeric polypeptide. The αCD3scFv/TF/αCD28scFvsingle-chain chimeric polypeptide was detected using ELISA with oneanti-TF monoclonal antibody for capture and a different anti-TFmonoclonal antibody for detection (FIG. 5 ). A purified tissue factorprotein with a similar concentration was used as a control.

A further in vitro experiment was performed to determine whether theαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide is capable ofactivating human peripheral blood mononuclear cells (PBMCs). Fresh humanleukocytes were obtained from the blood bank and peripheral bloodmononuclear cells (PBMC) were isolated using density gradient Histopaque(Sigma). The cells were counted and resuspended in 0.2×10⁶/mL in a96-well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco)supplemented with 2 mM L-glutamine (Thermo Life Technologies),penicillin (Thermo Life Technologies), streptomycin (Thermo LifeTechnologies), and 10% FBS (Hyclone)). The cells were stimulated withαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide from 0.01 nM to1000 nM for 3 days at 37° C., 5% CO₂. After 72 hours, the cells wereharvested and surface stained for CD4-488, CD8-PerCP Cy5.5, CD25-BV421,CD69-APCFire750, CD62L-PE Cy7, and CD44-PE (Biolegend) for 30 minutes.After surface staining, the cells were washed (1500 RPM for 5 minutes atroom temperature) in FACS buffer (1×PBS (Hyclone) with 0.5% BSA (EMDMillipore) and 0.001% sodium azide (Sigma)). After two washes, the cellswere resuspended in 300 μL of FACS buffer and analyzed by Flow Cytometry(Celesta-BD Bioscience). The data in FIGS. 6 and 7 show that theαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide is able tostimulate both CD8⁺ and CD4⁺ T-cells.

A further experiment was performed, in which PBMCs isolated from bloodusing Histopaque (Sigma) were counted and resuspended in 0.2×10⁶/mL in a96-well flat bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco)supplemented with 2 mM L-glutamine (Thermo Life Technologies),penicillin (Thermo Life Technologies), streptomycin (Thermo LifeTechnologies), and 10% FBS (Hyclone)). The cells were then stimulatedwith the αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide from0.01 nM to 1000 nM for 3 days at 37° C., 5% CO₂. After 72 hours, thecells were harvested and surface stained for CD4-488, CD8-PerCP Cy5.5,CD25-BV421, CD69-APCFire750, CD62L-PE Cy7, and CD44-PE (Biolegend) for30 minutes. After surface staining, the cells were washed (1500 RPM for5 minutes at room temperature) in FACS buffer (1×PBS (Hyclone) with 0.5%BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes,the cells were resuspended in 300 μL of FACS buffer and analyzed by FlowCytometry (Celesta-BD Bioscience). The data again show that theαCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide was able tostimulate activation of CD4⁺ T cells (FIG. 8 ).

Example 3. Production and Characterization of the Exemplary Single-ChainChimeric Polypeptide IL-2/TF/IL-2

An exemplary single-chain chimeric polypeptide including a firsttarget-binding domain that binds to an IL-2 receptor, a soluble humantissue factor domain, and a second target-binding domain that binds toan IL-2 receptor was generated (IL-2/TF/IL-2) (FIG. 9 ). The nucleicacid and amino acid sequences of this single-chain chimeric polypeptideare shown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide(IL-2/TF/IL-2) (SEQ ID NO: 111) (Signal peptide)ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC(First IL-2 fragment) GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACC (Human tissue factor 219 form)AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Second IL-2 fragment)GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACTExemplary Single-Chain Chimeric Polypeptide (IL-2/TF/IL-2)(SEQ ID NO: 110) (Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-2)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE(Human IL-2) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

The nucleic acid encoding IL-2/TF/IL-2 was cloned into a modifiedretrovirus expression vector as described previously (Hughes et al., HumGene Ther 16:457-72, 2005). The expression vector encoding IL-2/TF/IL-2was transfected into CHO-K1 cells. Expression of the expression vectorin CHO-K1 cells allowed for secretion of the soluble IL-2/TF/IL-2single-chain chimeric polypeptide (referred to as 2t2), which can bepurified by anti-TF antibody affinity and other chromatography methods.

IL-2 and IL-2/TF/IL-2 Promoted IL-2Rβ and Common γ Chain Containing 32DβCell Proliferation in a Similar Manner

To evaluate the IL-2 activity of IL-2/TF/IL-2, IL-2/TF/IL-2 was comparedwith recombinant IL-2 for promoting proliferation of 32Dβ cells thatexpress IL-2Rβ and common γ chain. IL-2 dependent 32Dβ cells were washed5 times with IMDM-10% FBS and seeded to the wells at 2×10⁴ cells/well.Serial dilutions of IL-2/TF/IL-2 or IL-2 were added to the cells (FIG.10 ). Cells were incubated in a CO₂ incubator at 37° C. for 3 days. Cellproliferation was detected by adding 10 μl of WST1 to each well on day 3and incubating for an additional 3 hours in a CO₂ incubator at 37° C.The amount of formazan dye produced was analyzed by measuring theabsorbance at 450 nm. As shown in FIG. 10 , IL-2/TF/IL-2 and IL-2activated 32Dβ cells in a similar manner. The EC₅₀ of IL-2/TF/IL-2 andIL-2 was 158.1 pM and 140 pM, respectively.

IL-2/TF/IL-2 Showed Improved Ability to Promote IL-2Rαβγ ContainingCTLL-2 Cell Proliferation as Compared to IL-2

To evaluate the IL-2 activity of IL-2/TF/IL-2, IL-2/TF/IL-2 was comparedwith recombinant IL-2 for promoting proliferation of CTLL-2 cells thatexpress IL-2Rα, IL-2Rβ and common γ chain. IL-2 dependent CTLL-2 cellswere washed 5 times with IMDM-10% FBS and seeded to the wells at 2×10⁴cells/well. Serial dilutions of IL-2/TF/IL-2 or IL-2 were added to thecells (FIG. 11 ). Cells were incubated in a CO₂ incubator at 37° C. for3 days. Cell proliferation was detected by adding 10 μl of WST1 to eachwell in the day 3 and incubating for an additional 3 hours in a CO₂incubator at 37° C. The amount of formazan dye produced was analyzed bymeasuring the absorbance at 450 nm. As shown in FIG. 3 , IL-2/TF/IL-2promoted CTLL-2 cell proliferation 4-5-fold stronger than IL-2. The EC₅₀of IL-2/TF/IL-2 was 123.2 pM and IL-2 was 548.2 pM.

IL-2/TF/IL-2 Suppressed the Increase of the High Fat-InducedHyperglycemia in ApoE^(−/−) Mice

Six-week-old female ApoE^(−/−) mice (Jackson Lab) were fed with standardchow diet or high diet fat containing 21% fat, 0.15% cholesterol, 34.1%sucrose, 19.5% casein, and 15% starch (TD88137, Harlan Laboratories) andmaintained in the standard conditions. At week 7, mice fed with high fatdiet were randomly assigned into the control group and treatment group.Mice then received either IL-2/TF/IL-2 (treatment group) or PBS (chowdiet group and control group) per subcutaneous injection at a dosage of3 mg/kg. Three days post dosing, the mice were fasted overnight, andblood samples were collected through retro-orbital venous plexuspuncture. Overnight fasting glucose levels were measured using aOneTouch Glucometer. As shown in FIG. 12 , the results showed thatIL-2/TF/IL-2 injection effectively suppresses the increase of glucoselevels in ApoE^(−/−) mice.

IL-2/TF/IL-2 Significantly Upregulate the Ratio of CD4⁺CD25⁺FoxP3⁺ TRegulatory (Treg) Cells in Blood Lymphocytes

Six-week-old female ApoE^(−/−) mice (Jackson Lab) were fed with standardchow diet or high diet fat containing 21% fat, 0.15% cholesterol, 34.1%sucrose, 19.5% casein, and 15% starch (TD88137, Harlan Laboratories) andmaintained in the standard conditions. At week 7, mice fed with the highfat diet were randomly assigned into control group and treatment group.Mice then received either IL-2/TF/IL-2 (treatment group) or PBS (chowdiet group and control group) per subcutaneous injection at a dosage of3 mg/kg. Three days after the dosing, overnight fasting blood sampleswere collected through retro-orbital venous plexus puncture andincubated with ACK lysing buffer (Thermo Fisher Scientific) at 37° C.for 5 minutes. Samples were then resuspended in FACS buffer (1×PBS(Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma))and surface stained with FITC-anti-CD4 and APC-anti-CD25 antibodies(BioLegend) for 30 minutes. Surface-stained samples were further fixedand premetallized with Fix/Perm buffer (BioLegend) and intracellularstained with PE-anti-Foxp3 antibody (BioLegend). After staining, cellswere washed twice with FACs buffer followed by centrifugation at 1500RPM for 5 minutes at room temperature. The cells were analyzed by flowcytometry (Celesta-BD Bioscience). As shown in FIG. 13 , IL-2/TF/IL-2treatment significantly increased Treg populations in blood lymphocytes(3.5%±0.32) compared to the untreated groups (0.4%±0.16 for chow dietgroup and 0.46%±0.09 for high fat diet group).

Purification Elution Chromatograph of IL-2/TF/IL-2 from Anti-TF AntibodyAffinity Column

IL-2/TF/IL-2 harvested from cell culture was loaded onto the anti-TFantibody affinity column equilibrated with 5 column volumes of PBS.After sample loading, the column was washed with 5 column volumes ofPBS, followed by elution with 6 column volumes of 0.1M acetic acid, pH2.9. A280 elution peak was collected and then neutralized to pH 7.5-8.0with 1M Tris base. The neutralized sample was then buffer exchanged intoPBS using Amicon centrifugal filters with a 30 kDa molecular weightcutoff. As shown in FIG. 14 , the anti-TF antibody affinity column boundto IL-2/TF/IL-2 which contains TF as a fusion domain. Thebuffer-exchanged protein sample was stored at 2-8° C. for furtherbiochemical analyses and biological activity tests. After each elution,the anti-TF antibody affinity column was stripped using 6 column volumesof 0.1M glycine, pH 2.5. The column was then neutralized using 5 columnvolumes of PBS, and 7 column volumes of 20% ethanol for storage. Theanti-TF antibody affinity column was connected to a GE Healthcare AKTAAvant system. The flow rate was 4 mL/min for all steps except for theelution step, which was 2 mL/min.

Analytical Size Exclusion Chromatography (SEC) Analysis of IL-2/TF/IL-2

To analyze IL-2/TF/IL-2 using analytical size exclusion chromatography(SEC), a Superdex 200 Increase 10/300 GL gel filtration column (from GEHealthcare) was connected to an AKTA Avant system (from GE Healthcare).The column was equilibrated with 2 column volumes of PBS. The flow ratewas 0.7 mL/min. A sample containing IL-2/TF/IL-2 in PBS was injectedinto the Superdex 200 column using a capillary loop, and analyzed bySEC. The SEC chromatograph of the sample is shown in FIG. 15 . The SECresults indicated two protein peaks for IL-2/TF/IL-2.

Reduced SDS PAGE of IL-2/TF/IL-2

To determine the purity and molecular weight of the protein,IL-2/TF/IL-2 protein sample purified with anti-TF antibody affinitycolumn was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12%NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method under reducedcondition. After electrophoresis, the gel was stained with InstantBluefor about 30 min, followed by destaining overnight in purified water.

To verify that the IL-2/TF/IL-2 protein undergoes glycosylation aftertranslation in CHO cells, a deglycosylation experiment was conductedusing the Protein Deglycosylation Mix II kit from New England Biolabsaccording to the manufacturer's instructions. FIGS. 16A and 16B show thereduced SDS-PAGE analysis of the sample in non-deglycosylated (lane 1 inred outline) and deglycosylated (lane 2 in yellow outline) state. Theresults show that the IL-2/TF/IL-2 protein is glycosylated whenexpressed in CHO cells. After deglycosylation, the purified sample ranwith expected molecular weights (56 kDa) in reduced SDS gel. Lane M wasloaded with 10 μL of SeeBlue Plus2 Prestained Standard.

In Vivo Characterization of IL-2/TF/IL-2

IL-2/TF/IL-2 was subcutaneously injected into C57BL/6 mice at variousdoses to determine the immunostimulatory activity of IL-2/TF/IL-2 invivo. Mice were subcutaneously treated with control solution (PBS) orIL-2/TF/IL-2 at 0.1, 0.4, 2 and 10 mg/kg. The treated mice wereeuthanized day 3 post treatment. The mouse spleens were collected andweighed day 3 post treatment. Single splenocyte suspensions wereprepared, and the prepared splenocytes were stained for CD4⁺ T cells,CD8⁺ T cells and NK cells (with fluorochrome-conjugated anti-CD4, -CD8,and -NK1.1 antibodies), and analyzed by flow cytometry. The resultsshowed that IL-2/TF/IL-2 was effective at expanding splenocytes based onspleen weight (FIG. 17A) especially at 0.1-10 mg/kg. The percentage ofCD8⁺ T cells were higher compared to control-treated mice (FIG. 17B) at2 and 10 mg/kg. The percentage of NK cells were higher compared tocontrol-treated mice (FIG. 17B) at all doses tested.

It has been known that IL-2 upregulates CD25 expression by immunocytes.We therefore accessed CD25 expression of CD4⁺ T cells, CD8⁺ T cells andNK cells in the IL-2/TF/IL-2 treated mice. C57BL/6 mice weresubcutaneously treated with IL-2/TF/IL-2 as described in the paragraphabove. The splenocytes were stained with fluorochrome-conjugatedanti-CD4, -CD8, CD25 and NK1.1 monoclonal antibodies. The CD25expression (MFI) of splenocyte subsets was analyzed by flow cytometry.As shown in FIG. 18 , at the doses and time point (day 3) tested,IL-2/TF/IL-2 significantly upregulated CD25 expression by CD4⁺ T cellsbut not CD8+ T cells or NK cells.

The pharmacokinetics of IL-2/TF/IL-2 in C57BL/6 mice was alsoinvestigated. IL-2/TF/IL-2 was subcutaneously injected into C57BL/6 miceat 1 mg/kg. The mouse blood was drawn from tail vein at various timepoints as shown in FIG. 19 and the serum was prepared. IL-2/TF/IL-2concentrations were determined with ELISA (Capture: anti-tissue factorantibody; Detection: biotinylated anti-human IL-2 antibody followed bySA-HRP and ABTS substrate). The half-life of IL-2/TF/IL-2 was 1.83 hourscalculated with PK Solutions 2.0 (Summit Research Services).

IL-2/TF/IL-2 Attenuated the Formation of High Fat-InducedAtherosclerotic Plaques in ApoE^(−/−) Mice

Six-week-old female ApoE^(−/−) mice (The Jackson Laboratory) were fedwith standard chow diet or high diet fat (21% fat, 0.15% cholesterol,34.1% sucrose, 19.5% casein, and 15% starch) (TD88137, HarlanLaboratories) and maintained in the standard conditions. At week 7, micefed with high fat diet (HFD) were randomly assigned into control groupand treatment group. Mice were then administrated either IL-2/TF/IL-2(treatment group) or PBS (chow diet group and control group)subcutaneously at a dosage of 3 mg/kg weekly for 4 weeks. At week 12,all mice were euthanized by isoflurane. Aortas were collected, openedlongitudinally and stained with Sudan IV solution (0.5%) using en facemethod. The percentage of plaque area (red color as shown in FIG. 20A)relative to total aorta area was then quantified with Image J software.FIG. 20A shows a representative view of atherosclerotic plaques fromeach group. FIG. 20B shows the results of quantitative analysis ofatherosclerotic plaques of each group. The percentage of plaque areas incontrol group (HF Diet) was much higher than the treatment group(HFD+IL-2/TF/IL-2), being 10.28% vs 4.68%.

IL-2/TF/IL-2 Suppresses the Progression of Type 2 Diabetes

Male BKS.Cg-Dock7^(m)+/+Lepr^(db)/J (db/db (Jackson Lab)) mice were fedwith standard chow diet and received drinking water ad libitum. At theage of six weeks, mice were randomly assigned into control group andtreatment group. The treatment group received IL-2/TF/IL-2 bysubcutaneous injection at 3 mg/kg bi-weekly, while control groupreceived vehicle (PBS) only. Overnight fasting glucose levels weremeasure weekly using a OneTouch Glucometer. The results showed thatIL-2/TF/IL-2 effectively suppressed the increase of glucose levels inBKS.Cg-Dock7^(m)+/+Lepr^(db)/J mice.

IL-2/TF/IL-2 Significantly Upregulates the Ratio of CD4⁺CD25⁺FoxP3⁺ TRegulatory Cells in Blood Lymphocytes after the First Injection

Male BKS.Cg-Dock7^(m)+/+Lepr^(db)/J (db/db) (The Jackson Laboratory)mice were fed with standard chow diet and received drinking water adlibitum. At the age of six weeks, mice were randomly assigned intocontrol group and treatment group. The treatment group receivedIL-2/TF/IL-2 by subcutaneous injection at 3 mg/kg bi-weekly, while thecontrol group received vehicle (PBS) only. Four days after the firstdrug injection, overnight fasting blood samples were collected andincubated with ACK lysing buffer (Thermo Fisher Scientific) at 37° C.for 5 minutes. Samples were then resuspended in FACS buffer (1×PBS(Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma))and surface stained with FITC-anti-CD4 and APC-anti-CD25 antibodies(BioLegend) for 30 minutes. Surface-stained samples were further fixedand premetallized with Fix/Perm buffer (BioLegend) and intracellularstained with PE-anti-Foxp3 antibody (BioLegend). After staining, cellswere washed twice with FACs buffer and were analyzed by flow cytometry(Celesta-BD Bioscience). The percentage of CD4⁺CD25⁺FoxP3⁺ Tregs inblood lymphocytes were measured. As shown in FIG. 22 , the resultsshowed that IL-2/TF/IL-2 significantly upregulated the ratio of Tregs inblood lymphocytes. * p<0.05

Example 4. Production and Characterization of the Exemplary Single-ChainChimeric Polypeptide IL-15/TF/IL-15

A second exemplary single-chain chimeric polypeptide including a firsttarget-binding domain that binds to an IL-15 receptor, a soluble humantissue factor domain, and a second target-binding domain that binds toan IL-15 receptor was generated (IL-15/TF/IL-15 or IL-15/TF/IL-15) (FIG.23 ). The nucleic acid and amino acid sequences of this single-chainchimeric polypeptide are shown below.

Nucleic Acid Encoding Exemplary Single-Chain Chimeric Polypeptide(IL-15/TF/IL-15) (SEQ ID NO: 117) (Signal peptide)ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC(First IL-15 fragment) AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTAGC (Human tissue factor 219 form)AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Second IL-15 fragment)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCCExemplary Single-Chain Chimeric Polypeptide (IL-15/TF/IL-15)(SEQ ID NO: 116) (Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-15)NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS(Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQE KGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS

The nucleic acid encoding IL-15/TF/IL-15 was cloned into a modifiedretrovirus expression vector as described previously (Hughes et al., HumGene Ther 16:457-72, 2005). The expression vector encodingIL-15/TF/IL-15 was transfected into CHO-K1 cells. Expression of theexpression vector in CHO-K1 cells allowed for secretion of the solubleIL-15/TF/IL-15 single-chain chimeric polypeptide (referred to as 15t15),which can be purified by anti-TF antibody affinity and otherchromatography methods.

IL-15/TF/IL-15 Promotes IL-2Rβ and Common γ Chain Containing 32Dβ CellProliferation

IL-15 activity of IL-15/TF/IL-15 was compared with recombinant IL-15 inIL2Rβ and common γ chain expressed 32Dβ cells. IL-15 dependent 32Dβcells were washed five times with IMDM-10% FBS and seeded to the wellsat 2×10⁴ cells/well. Serial dilutions of IL-15/TF/IL-15 or IL-15 wereadded to the cells (FIG. 24 ). Cells were incubated in a CO₂ incubatorat 37° C. for 3 days. Cell proliferation was detected by adding 10 μl ofWST1 to each well in the day 3 and incubating for an additional 3 hoursin a CO₂ incubator at 37° C. The amount of formazan dye produced wasanalyzed by measuring the absorbance at 450 nm. As shown in FIG. 24 ,IL-15/TF/IL-15 promoted 32Dβ cell proliferation less efficiently ascompared to IL-15. The EC₅₀ of IL-15/TF/IL-15 and IL-15 was 161.4 pM and1.6 pM. respectively.

Purification Elution Chromatograph of IL-15/TF/IL-15 from Anti-TFAntibody Affinity Column

IL-15/TF/IL-15 harvested from cell culture was loaded onto the anti-TFantibody affinity column equilibrated with 5 column volumes of PBS.After sample loading, the column was washed with 5 column volumes ofPBS, followed by elution with 6 column volumes of 0.1M acetic acid, pH2.9. A280 elution peak was collected and then neutralized to pH 7.5-8.0with 1M Tris base. The neutralized sample was then buffer exchanged intoPBS using Amicon centrifugal filters with a 30 kDa molecular weightcutoff. As shown in FIG. 25 , the anti-TF antibody affinity column boundto IL-15/TF/IL-15 which contains TF as a fusion domain. Thebuffer-exchanged protein sample was stored at 2-8° C. for furtherbiochemical analyses and biological activity tests. After each elution,the anti-TF antibody affinity column was stripped using 6 column volumesof 0.1M glycine, pH 2.5. The column was then neutralized using 5 columnvolumes of PBS, and 7 column volumes of 20% ethanol for storage. Theanti-TF antibody affinity column was connected to a GE Healthcare AKTAAvant system. The flow rate was 4 mL/min for all steps except for theelution step, which was 2 mL/min.

Reduced SDS-PAGE of IL-15/TF/IL-15

To determine the purity and molecular weight of the protein,IL-15/TF/IL-15 (15t15) protein sample purified with anti-TF antibodyaffinity column was analyzed by sodium dodecyl sulfate polyacrylamidegel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method underreduced condition. After electrophoresis, the gel was stained withInstantBlue for about 30 min, followed by destaining overnight inpurified water.

To verify that the IL-15/TF/IL-15 protein undergoes glycosylation aftertranslation in CHO cells, a deglycosylation experiment was conductedusing the Protein Deglycosylation Mix II kit from New England Biolabsand the manufacturer's instructions. FIGS. 26A and 26B show the reducedSDS-PAGE analysis of the sample in non-deglycosylated (lane 1 in redoutline) and deglycosylated (lane 2 in yellow outline) state. Theresults showed that the IL-15/TF/IL-15 protein is glycosylated whenexpressed in CHO cells. After deglycosylation, the purified sample ranwith expected molecular weights (50 kDa) in reduced SDS gel. Lane M wasloaded with 10 μL of SeeBlue Plus2 Prestained Standard.

Example 5: Stimulation of NK Cells In Vivo by IL-2/TF/IL-2 (2t2)

A set of experiments was performed to determine the effect of the 2t2construct on immune stimulation in C57BL/6 mice. In these experiments,C57BL/6 mice were subcutaneously treated with control solution (PBS) or2t2 at 0.1, 0.4, 2, and 10 mg/kg. Treated mice were euthanized 3 dayspost-treatment. Spleen weight was measured and single splenocytesuspensions were prepared. Splenocytes suspensions were stained withconjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies. Thepercentage of CD4⁺ T cells, CD8⁺ T cells, and NK cells, and CD25expression on lymphocyte subsets were analyzed by flow cytometry. FIG.27A shows that 2t2 was effective at expanding splenocytes based onspleen weight especially at a dose level of 0.1-10 mg/kg. Followingtreatment, the percentage of CD8⁺ T cells were higher in 2t2-treatedmice compared to control-treated mice at 2 and 10 mg/kg (FIG. 27B). Thepercentage of NK cells were also higher in 2t2-treated mice compared tocontrol-treated mice at all doses of 2t2 tested (FIG. 27B).Additionally, 2t2 significantly upregulated CD25 expression by CD4⁺ Tcells, but not CD8⁺ T cells and NK cells following treatment at 0.4 to10 mg/kg (FIG. 27C).

A set of experiments was performed to determine the effect of the 2t2construct on immune stimulation in ApoE^(−/−) mice fed with a Westerndiet. In these experiments, 6-week old female B6.129P2-ApoE^(tm1Unc)/Jmice (Jackson Laboratory) were fed with a Western diet containing 21%fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch(TD88137, Envigo Laboratories). After 8-weeks of the Western diet, themice were injected subcutaneously with 2t2 at 3 mg/kg. Three days posttreatment, mice were fasted for 16 hours and then blood samples werecollected through retro-orbital venous plexus puncture. The blood wasmixed with 10 μL 0.5 M EDTA, and 20 μL blood was taken for lymphocytesubsets analysis. The red blood cells were lysed with ACK (0.15 M NH₄Cl,1.0 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.4) and the lymphocytes were stainedwith anti-mouse CD8a and anti-mouse NK1.1 antibodies for 30 minutes at4° C. in FACS staining buffer (1% BSA in PBS). The cells were washedonce and analyzed with a BD FACS Celesta. For Treg staining, ACK treatedblood lymphocytes were stained with anti-mouse CD4 and anti-mouse CD25antibodies for 30 minutes at 4° C. in FACS staining buffer. The cellswere washed once and resuspended in fixation/permeabilization workingsolution and incubated at room temperature for 60 minutes. The cellswere washed once and resuspended in permeabilization buffer. The sampleswere centrifuged at 300-400×g for 5 minutes at room temperature and thesupernatant was then discarded. The cell pellet was resuspended inresidual volume and the volume adjusted to about 100 μL with 1×permeabilization buffer. Anti-Foxp3 antibody was added to the cells, andthe cells were incubated for 30 minutes at room temperature.Permeabilization buffer (200 μL) was added to the cells, and the cellswere centrifuged at 300-400×g for 5 minutes at room temperature. Thecells were resuspended in flow cytometry staining buffer and analyzed ona flow cytometer. FIGS. 28B-28C show that treatment with 2t2 increasedthe percentage of NK cells and CD8⁺ T cells in ApoE^(−/−) mice fed withWestern diet. FIG. 28A shows that treatment with 2t2 also increased thepercentage of Treg cells.

Example 6: Induction of Proliferation of Immune Cells In Vivo

A set of experiments was performed to determine the effect of the 2t2construct on immune cell stimulation in C57BL/6 mice. In theseexperiments, C57BL/6 mice were subcutaneously treated with controlsolution (PBS) or 2t2 at 0.1, 0.4, 2, and 10 mg/kg. Treated mice wereeuthanized 3 days post-treatment. Spleen weight was measured and singlesplenocyte suspensions were prepared. The splenocyte suspensions werestained with conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK)antibodies. The percentage of CD4⁺ T cells, CD8⁺ T cells, and NK cellswere analyzed by flow cytometry. FIG. 29A shows that 2t2 treatment waseffective at expanding splenocytes based on spleen weight especially at0.1-10 mg/kg. The percentage of CD8⁺ T cells was higher compared tocontrol-treated mice at 2 and 10 mg/kg (FIG. 29B). The percentage of NKcells was higher compared to control-treated mice at all doses of 2t2tested (FIG. 29B). These results demonstrate that 2t2 treatment was ableto induce proliferation of CD8⁺ T cells and NK cells in C57BL/6 mice.

A set of experiments was performed to determine the effect of the 2t2construct on immune stimulation in ApoE^(−/−) mice fed with a Westerndiet. In these experiments, 6-week old female B6.129P2-ApoE^(tm1Unc)/Jmice (Jackson Laboratory) were fed with a Western diet containing 21%fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch(TD88137, Envigo Laboratories). After 8-week of the Western diet, themice were injected subcutaneously with 2t2 at 3 mg/kg. Three dayspost-treatment, the mice were fasted for 16 hours and then blood sampleswere collected through retro-orbital venous plexus puncture. The bloodwas mixed with 10 μL 0.5 M EDTA and 20 μL blood was taken for lymphocytesubsets analysis. The red blood cells were lysed with ACK (0.15 M NH₄Cl,1.0 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.4) and the lymphocytes were stainedwith anti-mouse CD8a and anti-mouse NK1.1 antibodies for 30 minutes at4° C. in FACS staining buffer (1% BSA in PBS). The cells were washedonce and resuspended in Fixation Buffer (BioLegend Cat #420801) for 20minutes at room temperature. The cells were centrifuged at 350×g for 5minutes, the fixed cells were resuspended in Intracellular StainingPermeabilization Wash Buffer (BioLegend Cat #421002) and thencentrifuged at 350×g for 5 minutes. The cells were then stained withanti-Ki67 antibody for 20 minutes at RT. The cells were washed twicewith Intracellular Staining Permeabilization Wash Buffer and centrifugedat 350×g for 5 minutes. The cells were then resuspended in FACS stainingbuffer. Lymphocyte subsets were analyzed with a BD FACS Celesta. FIGS.30A and 30B shows treatment of ApoE^(−/−) mice with 2t2 also inducedproliferation (Ki67-positive staining) in NK and CD8⁺ T cells.

Example 7: Treatment of Diabetes

A set of experiments was performed to investigate amelioration ofWestern diet-induced hyperglycemia in ApoE^(−/−) mice by 2t2. In theseexperiments, 6-week old female B6.129P2-ApoE^(tm1Unc)/J mice (JacksonLaboratory) were fed with a Western diet containing 21% fat, 0.15%cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137,Envigo Laboratories). After 8-weeks of the Western diet, the mice wereinjected subcutaneously with 2t2 at 3 mg/kg. Three days post-treatment,the mice were fasted for 16 hours and then blood samples were collectedthrough retro-orbital venous plexus puncture. Blood glucose was detectedwith a glucose meter (OneTouch UltraMini) and GenUltimated test stripsusing a drop of fresh blood. As shown in FIG. 31A, 2t2 treatmentsignificantly reduced hyperglycemia induced by the Western diet(p<0.04). The plasma insulin and resistin levels were analyzed withMouse Rat Metabolic Array by Eve Technologies. HOMA-IR was calculatedusing the following formula: homeostatic model assessment-insulinresistance=Glucose (mg/dL)*Insulin (mU/mL)/405. As shown in FIG. 31B,2t2 treatment reduced insulin resistance compared to the untreatedgroup. 2t2 (p<0.02) reduced resistin levels significantly compared tothe untreated group as shown in FIG. 31C, which may relate to thereduced insulin resistance induced by 2t2 (FIG. 31B).

Example 8. Upregulation of CD44 Memory T Cells

C57BL/6 mice were subcutaneously treated with 2t2. The treated mice wereeuthanized and the single splenocyte suspensions were prepared 4 days(TGFRt15-TGFRs) or 3 days (2t2) following the treatment. The preparedsplenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8and anti-CD44 antibodies and the percentages of CD44^(high) T cells inCD4⁺ T cells or CD8⁺ T cells were analyzed by flow cytometry. Theresults show that 2t2 upregulated expression of the memory marker CD44on CD4⁺ and CD8⁺ T cells (FIG. 32 ). These findings indicate that 2t2was able to induce mouse T cells to differentiate into memory T cells.

Example 9. Tissue Factor Coagulation Assays Following Treatment withSingle-Chain or Multi-Chain Chimeric Polypeptides

A set of experiments was performed to assess blood coagulation followingtreatment with single-chain or multi-chain chimeric polypeptides. Toinitiate the blood coagulation cascade pathway, tissue factor (TF) bindsto Factor VIIa (FVIIa) to form a TF/FVIIa complex. The TF/FVIIa complexthen binds Factor X (FX) and converts FX to FXa.

An assay to measure blood coagulation involves measuring activation ofFactor X (FX). Briefly, TF/VIIa activates blood coagulation Factor X(FX) to Factor Xa (FXa) in the presence of calcium and phospholipids.TF₂₄₃, which contains the transmembrane domain of TF, has much higheractivity in activating FX to FXa than TF219, which does not contain thetransmembrane domain. TF/VIIa dependent activation of FX is determinedby measuring FXa activity using an FXa-specific chromogenic substrateS-2765 (Diapharma, West Chester, Ohio). The color change of S-2765 canbe monitored spectrophotometrically and is proportional to theproteolytic activity of FXa.

In these experiments, FX activation with a multi-chain chimericpolypeptide (18t15-12s, mouse (m)21t15, 21t15-TGFRs, and 21t15-7s) wascompared with a positive control (Innovin) or TF₂₁₉. TF₂₁₉ (orTF₂₁₉-containing multi-chain chimeric polypeptides)/FVIIa complexes weremixed at an equal molar concentration (0.1 nM each) in a volume of 50 μLin round bottom wells of a 96-well ELISA plate, after which 10 μL of 180nM FX was added. After 15 minutes of incubation at 37° C., during whichtime FX was converted to FXa, 8 μL of 0.5 M EDTA (which chelates calciumand thus terminates FX activation by TF/VIIa) was added to each well tostop FX activation. Next, 10 μL of 3.2 mM S-2765 substrate was added tothe reaction mixture. Immediately, the plate absorbance was measured at405 nm and was recorded as the absorbance at time 0. The plate was thenincubated for 10-20 minutes at 37° C. The color change was monitored byreading absorbance at 405 nm following the incubation. Results of FXactivation as measured by FXa activity using chromogenic substrateS-2765 are shown in FIG. 33 . In this experiment, Innovin, which is acommercial prothrombin reagent containing lipidated recombinant humanTF243, was used as a positive control for FX activation. Innovin wasreconstituted with purified water to about 10 nM of TF243. Next, 0.1 nMTF/VIIa complex was made by mixing an equal volume of 0.2 nM of FVIIawith 0.2 nM of Innovin. Innovin demonstrated very potent FX activationactivity, while TF219 and TF219-containing multi-chain chimericpolypeptides had very low FX activation activity, confirming that TF219is not active in a TF/FVIIa complex for activating natural substrate FXin vivo.

Example 10: Induction of Treg Cells by 2t2

The peripheral blood mononuclear cells (PBMC) of a heathy donor (Donor163) were isolated from 5 mL of whole blood buffy coats by Ficoll PaquePlus (GE17144003). The PBMC were then lysed with ACK to remove red bloodcells. Cells were washed with IMDM-10% FBS and counted. 1.8×10⁶ cells(100 μL/tube) were seeded to the flow tubes and incubated with 50 μL ofdescending 2t2 or IL2 (15000, 1500, 150, 15, 1.5, 0.15, or 0 pM) and 50μL of pre-staining antibodies (anti-CD8-BV605 and anti-CD127-AF647).Cells were incubated for 30 min at 37° C. in water bath. 200 μL ofpre-warmed BD Phosflow Fix Buffer I (Cat #557870, Becton DickinsonBiosciences) was added for 10 min at 37° C. in water bath to stop thestimulation. Cells (4.5×10⁵ cells/100 μL) were transferred to a V-shape96-well plate and were spun down followed by permeabilization with 100μL of −20° C. pre-cooled BD Phosflow Perm Buffer III (Cat #BDBiosciences) for 30 min on ice. The cells were then extensively washed×2 with 200 μL of FACS buffer and stained with a panel of fluorescentantibodies (anti-CD25-PE, CD4-PerCP-Cy5.5, CD56-BV421, CD45RA-PE-Cy7 andpSTAT5a-AF488) to distinguish between different lymphocytesubpopulations and evaluate the pSTAT5a status. Cells were spun down andresuspended in 200 μL of FACS buffer for FACSCelesta analysis. As shownin Figure C1A, 6 pM of 2t2 was sufficient to induce the phosphorylationof Stat5a in CD4⁺CD25^(hi) T_(reg) cells while 43.11 pM of IL-2 wasrequired to induce phosphorylation of Stat5a in the same population oflymphocytes. In contrast, 2t2 was less active (FIG. 34B) or equallyactive (FIG. 34C) as compared to IL2 in inducing phosphorylation ofStat5a in CD4⁺CD25⁻T_(con) and CD8⁺ T_(con) cells. These results suggestthat 2t2 is superior as compared to IL2 in activating T_(reg) in humanPBMC, and that 2t2 demonstrates increased T_(reg) selectivity comparedto IL-2 in human blood lymphocyte pStat5a responses.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

A. EXEMPLARY EMBODIMENTS

Embodiment A1. A single-chain chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a second target-binding domain.

Embodiment A2. The single-chain chimeric polypeptide of embodiment A1,wherein the first target-binding domain and the soluble tissue factordomain directly abut each other.

Embodiment A3. The single-chain chimeric polypeptide of embodiment A1,wherein the single-chain chimeric polypeptide further comprises a linkersequence between the first target-binding domain and the soluble tissuefactor domain.

Embodiment A4. The single-chain chimeric polypeptide of any one ofembodiments A1-A3, wherein the soluble tissue factor domain and thesecond target-binding domain directly abut each other.

Embodiment A5. The single-chain chimeric polypeptide of any one ofembodiments A1-A3, wherein the single-chain chimeric polypeptide furthercomprises a linker sequence between the soluble tissue factor domain andthe second target-binding domain.

Embodiment A6. The single-chain chimeric polypeptide of embodiment A1,wherein the first target-binding domain and the second target-bindingdomain directly abut each other.

Embodiment A7. The single-chain chimeric polypeptide of embodiment A1,wherein the single-chain chimeric polypeptide further comprises a linkersequence between the first target-binding domain and the secondtarget-binding domain.

Embodiment A8. The single-chain chimeric polypeptide of embodiment A6 orA7, wherein the second target-binding domain and the soluble tissuefactor domain directly abut each other.

Embodiment A9. The single-chain chimeric polypeptide of embodiment A6 orA7, wherein the single-chain chimeric polypeptide further comprises alinker sequence between the second target-binding domain and the solubletissue factor domain.

Embodiment A10. The single-chain chimeric polypeptide of any one ofembodiments A1-A9, wherein the first target-binding domain and thesecond target-binding domain bind specifically to the same antigen.

Embodiment A11. The single-chain chimeric polypeptide of embodiment A10,wherein the first target-binding domain and the second target-bindingdomain bind specifically to the same epitope.

Embodiment A12. The single-chain chimeric polypeptide of embodiment A11,wherein the first target-binding domain and the second target-bindingdomain comprise the same amino acid sequence.

Embodiment A13. The single-chain chimeric polypeptide of any one ofembodiments A1-A9, wherein the first target-binding domain and thesecond target-binding domain bind specifically to different antigens.

Embodiment A14. The single-chain chimeric polypeptide of any one ofembodiments A1-A13, wherein one or both of the first target-bindingdomain and the second target-binding domain is an antigen-bindingdomain.

Embodiment A15. The single-chain chimeric polypeptide of embodiment A14,wherein the first target-binding domain and the second target-bindingdomain are each an antigen-binding domain.

Embodiment A16. The single-chain chimeric polypeptide of embodiment A13,wherein antigen-binding domain comprises a scFv or a single domainantibody.

Embodiment A17. The single-chain chimeric polypeptide of any one ofembodiments A1-A16, wherein one or both of the first target-bindingdomain and the second target-binding domain bind to a target selectedfrom the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22,CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3,CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200,IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA,B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR,DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-β receptorII (TGF-βRII), a ligand of TGF-βRIII, a ligand of DNAM-1, a ligand ofNKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligandfor a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptorfor IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7,a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, areceptor for IL-15, a receptor for IL-17, a receptor for IL-18, areceptor for IL-21, a receptor for PDGF-DD, a receptor for stem cellfactor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand(FLT3L), a receptor for MICA, a receptor for MICB, a receptor for aULP16-binding protein, a receptor for CD155, a receptor for CD122, and areceptor for CD28.

Embodiment A18. The single-chain chimeric polypeptide of any one ofembodiments A1-A16, wherein one or both of the first target-bindingdomain and the second target-binding domain is a soluble interleukin, asoluble cytokine protein, or a soluble cell surface protein.

Embodiment A19. The single-chain chimeric polypeptide of embodiment A18,wherein the soluble interleukin, solublecytokine protein, or solublecell surface protein is selected from the group consisting of: IL-1,IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment A20. The single-chain chimeric polypeptide of any one ofembodiments A1-A16, wherein one or both of the first target-bindingdomain and the second target-binding domain is a soluble interleukinreceptor, a soluble cytokine receptor, or a soluble cell surfacereceptor.

Embodiment A21. The single-chain chimeric polypeptide of embodiment A20,wherein the soluble interleukin receptor, soluble cytokine receptor, orsoluble cell surface receptor is a soluble TGF-βreceptor II (TGF-βRII),a soluble TGF-βRIII, a soluble NKG2D, a soluble NK30, a soluble NKp44, asoluble NKp46, a soluble DNAM1, a scMHCI, a scMHCII, a scTCR, a solubleCD155, or a soluble CD28.

Embodiment A22. The single-chain chimeric polypeptide of any one ofembodiments A1-A21, wherein the soluble tissue factor domain is asoluble human tissue factor domain.

Embodiment A23. The single-chain chimeric polypeptide of embodiment A22,wherein the soluble human tissue factor domain comprises a sequence thatis at least 80% identical to SEQ ID NO: 9.

Embodiment A24. The single-chain chimeric polypeptide of embodiment A23,wherein the soluble human tissue factor domain comprises a sequence thatis at least 90% identical to SEQ ID NO: 9.

Embodiment A25. The single-chain chimeric polypeptide of embodiment A24,wherein the soluble human tissue factor domain comprises a sequence thatis at least 95% identical to SEQ ID NO: 9.

Embodiment A26. The single-chain chimeric polypeptide of any one ofembodiments A22-A25, wherein the soluble human tissue factor domain doesnot comprise one or more of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment A27. The single-chain chimeric polypeptide of embodiment A26,wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment A28. The single-chain chimeric polypeptide of any one ofembodiments A1-A27, wherein the soluble tissue factor domain is notcapable of binding Factor VIIa.

Embodiment A29. The single-chain chimeric polypeptide of any one ofembodiments A1-A28, wherein the soluble tissue factor domain does notconvert inactive Factor X into Factor Xa.

Embodiment A30. The single-chain chimeric polypeptide of any one ofembodiments A1-A29, wherein the single-chain chimeric polypeptide doesnot blood stimulate coagulation in a mammal.

Embodiment A31. The single-chain chimeric polypeptide of any one ofembodiments A1-A30, wherein the single-chain chimeric polypeptidefurther comprises one or more additional target-binding domains at itsN- and/or C-terminus.

Embodiment A32. The single-chain chimeric polypeptide of embodiment A31,wherein the single-chain chimeric polypeptide comprises one or moreadditional target-binding domains at its N-terminus.

Embodiment A33. The single-chain chimeric polypeptide of embodiment A32,wherein one or more additional target-binding domains directly abuts thefirst target-binding domain, the second target-binding domain, or thesoluble tissue factor domain.

Embodiment A34. The single-chain chimeric polypeptide of embodiment A33,wherein the single-chain chimeric polypeptide further comprises a linkersequence between one of the at least one additional target-bindingdomains and the first target-binding domain, the second target-bindingdomain, or the soluble tissue factor domain.

Embodiment A35. The single-chain chimeric polypeptide of embodiment A31,wherein the single-chain chimeric polypeptide comprises one or moreadditional target-binding domains at its C-terminus.

Embodiment A36. The single-chain chimeric polypeptide of embodiment A35,wherein one of the one or more additional target-binding domainsdirectly abuts the first target-binding domain, the secondtarget-binding domain, or the soluble tissue factor domain.

Embodiment A37. The single-chain chimeric polypeptide of embodiment A35,wherein the single-chain chimeric polypeptide further comprises a linkersequence between one of the at least one additional target-bindingdomains and the first target-binding domain, the second target-bindingdomain, or the soluble tissue factor domain.

Embodiment A38. The single-chain chimeric polypeptide of embodiment A31,wherein the single-chain chimeric polypeptide comprises one or moreadditional target binding domains at its N-terminus and the C-terminus.

Embodiment A39. The single-chain chimeric polypeptide of embodiment A38,wherein one of the one or more additional antigen binding domains at theN-terminus directly abuts the first target-binding domain, the secondtarget-binding domain, or the soluble tissue factor domain.

Embodiment A40. The single-chain chimeric polypeptide of embodiment A38,wherein the single-chain chimeric polypeptide further comprises a linkersequence between one of the one or more additional antigen-bindingdomains at the N-terminus and the first target-binding domain, thesecond target-binding domain, or the soluble tissue factor domain.

Embodiment A41. The single-chain chimeric polypeptide of embodiment A38,wherein one of the one or more additional antigen binding domains at theC-terminus directly abuts the first target-binding domain, the secondtarget-binding domain, or the soluble tissue factor domain.

Embodiment A42. The single-chain chimeric polypeptide of embodiment A38,wherein the single-chain chimeric polypeptide further comprises a linkersequence between one of the one or more additional antigen-bindingdomains at the C-terminus and the first target-binding domain, thesecond target-binding domain, or the soluble tissue factor domain.

Embodiment A43. The single-chain chimeric polypeptide of any one ofembodiments A31-A42, wherein two or more of the first target-bindingdomain, the second target-binding domain, and the one or more additionaltarget-binding domains bind specifically to the same antigen.

Embodiment A44. The single-chain chimeric polypeptide of embodiment A43,wherein two or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains bind specifically to the same epitope.

Embodiment A45. The single-chain chimeric polypeptide of embodiment A44,wherein two or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains comprise the same amino acid sequence.

Embodiment A46. The single-chain chimeric polypeptide of embodiment A43,wherein the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains each bindspecifically to the same antigen.

Embodiment A47. The single-chain chimeric polypeptide of embodiment A46,wherein the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains each bindspecifically to the same epitope.

Embodiment A48. The single-chain chimeric polypeptide of embodiment A47,wherein the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains eachcomprise the same amino acid sequence.

Embodiment A49. The single-chain chimeric polypeptide of any one ofembodiments A31-A42, wherein the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains bind specifically to different antigens.

Embodiment A50. The single-chain chimeric polypeptide of any one ofembodiments A31-A49, wherein one or more of the first target-bindingdomain, the second target-binding domain, and the one or moretarget-binding domains is an antigen-binding domain.

Embodiment A51. The single-chain chimeric polypeptide of embodiment A50,wherein the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains are eachan antigen-binding domain.

Embodiment A52. The single-chain chimeric polypeptide of embodiment A51,wherein antigen-binding domain comprises a scFv or a single domainantibody.

Embodiment A53. The single-chain chimeric polypeptide of any one ofembodiments A31-A52, wherein one or more of the first target-bindingdomain, the second target-binding domain, and the one or moretarget-binding domains bind specifically to a target selected from thegroup consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123,IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4,MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R,MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3,EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4,TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-β receptor II(TGF-βRII), a ligand of TGF-βRIII, a ligand of DNAM-1, a ligand ofNKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligandfor a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptorfor IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7,a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, areceptor for IL-15, a receptor for IL-17, a receptor for IL-18, areceptor for IL-21, a receptor for PDGF-DD, a receptor for stem cellfactor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand(FLT3L), a receptor for MICA, a receptor for MICB, a receptor for aULP16-binding protein, a receptor for CD155, a receptor for CD122, and areceptor for CD28.

Embodiment A54. The single-chain chimeric polypeptide of any one ofembodiments A31-A52, wherein one or more of the first target-bindingdomain, the second target-binding domain, and the one or more additionaltarget-binding domains is a soluble interleukin, a soluble cytokineprotein, or a soluble cell surface protein.

Embodiment A55. The single-chain chimeric polypeptide of embodiment A54,wherein the soluble interleukin, soluble cytokine protein, or solublecell surface protein is selected from the group consisting of: IL-1,IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21,PDGF-DD, SCF, FLT3L, MICA, MICB, and a ULP16-binding protein.

Embodiment A56. The single-chain chimeric polypeptide of any one ofembodiments A31-A52, wherein one or more of the first target-bindingdomain, the second target-binding domain, and the one or more additionaltarget-binding domains is a soluble interleukin receptor, asolublecytokine receptor, or a soluble cell surface receptor.

Embodiment A57. The single-chain chimeric polypeptide of embodiment A56,wherein the soluble receptor is a soluble TGF-β receptor II (TGF-βRII),a soluble TGF-βRIII, a soluble NKG2D, a soluble NK30, a soluble NKp44, asoluble NKp46, a soluble DNAM1, a scMHCI, a scMHCII, a scTCR, a solubleCD155, a soluble CD122, a soluble CD3, or a soluble CD28.

Embodiment A58. The single-chain chimeric polypeptide of any one ofembodiments A1-A57, wherein the single-chain chimeric polypeptidefurther comprises a signal sequence at its N-terminal end.

Embodiment A59. The single-chain chimeric polypeptide of any one ofembodiments A1-A58, wherein the single-chain chimeric polypeptidefurther comprises a peptide tag positioned at the N-terminal end or theC-terminal end of the single-chain chimeric polypeptide.

Embodiment A60. A composition comprising any of the single-chainchimeric polypeptides of embodiments A1-A59.

Embodiment A61. The composition of embodiment A60, wherein thecomposition is a pharmaceutical composition.

Embodiment A62. A kit comprising at least one dose of the composition ofembodiment A60 or A61.

Embodiment A63. A method of stimulating an immune cell, the methodcomprising:

contacting an immune cell with an effective amount of any of thesingle-chain chimeric polypeptides of embodiments A1-A59 or thecomposition of embodiment A60 or A61.

Embodiment A64. The method of embodiment A63, wherein the immune cell iscontacted in vitro.

Embodiment A65. The method of embodiment A64, wherein the immune cellwas previously obtained from a subject.

Embodiment A66. The method of embodiment A65, wherein the method furthercomprises obtaining the immune cell from the subject prior to thecontacting step.

Embodiment A67. The method of embodiment A63, wherein the immune cell iscontacted in vivo.

Embodiment A68. The method of any one of embodiments A63-A67, whereinthe immune cell is selected from the group consisting of: an immaturethymocyte, a peripheral blood lymphocyte, a naïve T cell, a pluripotentTh cell precursor, a lymphoid progenitor cell, a Treg cell, a memory Tcell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, aTh3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ Tcell, a CD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage,a neutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment A69. The method of any one of embodiments A63-A68, whereinthe immune cell has previously been genetically modified to express achimeric antigen receptor or a recombinant T-cell receptor.

Embodiment A70. The method of any one of embodiments A63-A68, whereinthe method further comprises, after the contacting step, introducinginto the immune cell a nucleic acid encoding a chimeric antigen-receptoror a recombinant T-cell receptor.

Embodiment A71. The method of any one of embodiments A63-A70, whereinthe method further comprises administering the immune cell to a subjectin need thereof.

Embodiment A72. The method of embodiment A71, wherein the subject hasbeen identified or diagnosed as having an age-related disease orcondition.

Embodiment A73. The method of embodiment A72, wherein the age-relateddisease or condition is selected from the group consisting of:Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in panceatitis,glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatorybowel disease, intervertebral disc degeneration, macular degeneration,osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction.

Embodiment A74. The method of embodiment A71, wherein the subject hasbeen identified or diagnosed as having a cancer.

Embodiment A75. The method of embodiment A74, wherein the cancer isselected from the group consisting of: solid tumor, hematological tumor,sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-celllymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lungcancer, renal cell carcinoma, gastic and esophageal cancer, pancreaticcancer, prostate cancer, breast cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, squamous cell head and neckcarcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma.

Embodiment A76. The method of embodiment A71, wherein the subject hasbeen diagnosed or identified as having an infectious disease.

Embodiment A77. The method of embodiment A76, wherein the infectiousdisease is infection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment A78. A method of inducing or increasing proliferation of animmune cell, the method comprising:

contacting an immune cell with an effective amount of any of thesingle-chain chimeric polypeptides of embodiments A1-A59 or thecomposition of embodiment A60 or A61.

Embodiment A79. The method of embodiment A88, wherein the immune cell iscontacted in vitro.

Embodiment A80. The method of embodiment A79, wherein the immune cellwas previously obtained from a subject.

Embodiment A81. The method of embodiment A80, wherein the method furthercomprises obtaining the immune cell from the subject prior to thecontacting step.

Embodiment A82. The method of embodiment A78, wherein the immune cell iscontacted in vivo.

Embodiment A83. The method of any one of embodiments A78-A82, whereinthe immune cell is selected from the group consisting of: an immaturethymocyte, a peripheral blood lymphocyte, a naïve T cell, a pluripotentTh cell precursor, a lymphoid progenitor cell, a Treg cell, a memory Tcell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, aTh3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ Tcell, a CD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage,a neutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment A84. The method of any one of embodiments A78-A83, whereinthe immune cell has previously been genetically modified to express achimeric antigen receptor or a recombinant T-cell receptor.

Embodiment A85. The method of any one of embodiments A78-A83, whereinthe method further comprises, after the contacting step, introducinginto the immune cell a nucleic acid encoding a chimeric antigen-receptoror a recombinant T-cell receptor.

Embodiment A86. The method of any one of embodiments A78-A85, whereinthe method further comprises administering the immune cell to a subjectin need thereof.

Embodiment A87. The method of embodiment A86, wherein the subject hasbeen identified or diagnosed as having an age-related disease orcondition.

Embodiment A88. The method of embodiment A87, wherein the age-relateddisease or condition is selected from the group consisting of:Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in panceatitis,glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatorybowel disease, intervertebral disc degeneration, macular degeneration,osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction.

Embodiment A89. The method of embodiment A86, wherein the subject hasbeen identified or diagnosed as having a cancer.

Embodiment A90. The method of embodiment A89, wherein the cancer isselected from the group consisting of: solid tumor, hematological tumor,sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-celllymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lungcancer, renal cell carcinoma, gastic and esophageal cancer, pancreaticcancer, prostate cancer, breast cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, squamous cell head and neckcarcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma.

Embodiment A91. The method of embodiment A86, wherein the subject hasbeen diagnosed or identified as having an infectious disease.

Embodiment A92. The method of embodiment A86, wherein the infectiousdisease is infection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment A93. A method of inducing differentiation of an immune cellinto a memory or memory-like immune cell, the method comprising:

contacting an immune cell with an effective amount of any of thesingle-chain chimeric polypeptides of embodiments A1-A59 or thecomposition of embodiment A60 or A61.

Embodiment A94. The method of embodiment A93, wherein the immune cell iscontacted in vitro.

Embodiment A95. The method of embodiment A94, wherein the immune cellwas previously obtained from a subject.

Embodiment A96. The method of embodiment A95, wherein the method furthercomprises obtaining the immune cell from the subject prior to thecontacting step.

Embodiment A97. The method of embodiment A93, wherein the immune cell iscontacted in vivo.

Embodiment A98. The method of any one of embodiments A93-A97, whereinthe immune cell is selected from the group consisting of: an immaturethymocyte, a peripheral blood lymphocyte, a naïve T cell, a pluripotentTh cell precursor, a lymphoid progenitor cell, a Treg cell, a Th17 cell,a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell, γδ T cell,an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell,a natural killer T cell, a mast cell, a macrophage, a neutrophil, adendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment A99. The method of any one of embodiments A93-A98, whereinthe immune cell has previously been genetically modified to express achimeric antigen receptor or a recombinant T-cell receptor.

Embodiment A100. The method of any one of embodiments A93-A98, whereinthe method further comprises, after the contacting step, introducinginto the immune cell a nucleic acid encoding a chimeric antigen-receptoror a recombinant T-cell receptor.

Embodiment A101. The method of any one of embodiments A93-A100, whereinthe method further comprises administering the immune cell to a subjectin need thereof.

Embodiment A102. The method of embodiment A101, wherein the subject hasbeen identified or diagnosed as having an age-related disease orcondition.

Embodiment A103. The method of embodiment A102, wherein the age-relateddisease or condition is selected from the group consisting of:Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in panceatitis,glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatorybowel disease, intervertebral disc degeneration, macular degeneration,osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction.

Embodiment A104. The method of embodiment A101, wherein the subject hasbeen identified or diagnosed as having a cancer.

Embodiment A105. The method of embodiment A104, wherein the cancer isselected from the group consisting of: solid tumor, hematological tumor,sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-celllymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lungcancer, renal cell carcinoma, gastic and esophageal cancer, pancreaticcancer, prostate cancer, breast cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, squamous cell head and neckcarcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma.

Embodiment A106. The method of embodiment A101, wherein the subject hasbeen diagnosed or identified as having an infectious disease.

Embodiment A107. The method of embodiment A106, wherein the infectiousdisease is infection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment A108. A method of killing a cancer cell, an infected cell, ora senescent cell in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anyof the single-chain chimeric polypeptides of embodiments A1-A59 or thecomposition of embodiment A60 or A61

Embodiment A109. The method of embodiment A108, wherein the subject hasbeen identified or diagnosed as having a cancer.

Embodiment A110. The method of embodiment A109, wherein the cancer isselected from the group consisting of: solid tumor, hematological tumor,sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-celllymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lungcancer, renal cell carcinoma, gastic and esophageal cancer, pancreaticcancer, prostate cancer, breast cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, squamous cell head and neckcarcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma.

Embodiment A111. The method of embodiment A108, wherein the subject hasbeen identified or diagnosed as having an aging-related disease orcondition.

Embodiment A112. The method of embodiment A111, wherein theaging-related disease or condition is selected from the group consistingof: Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpanceatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction.

Embodiment A113. A method of treating a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of any of the single-chain chimeric polypeptides ofembodiments A1-A59 or the composition of embodiment A60 or A61

Embodiment A114. The method of embodiment A113, wherein the subject hasbeen identified or diagnosed as having a cancer.

Embodiment A115. The method of embodiment A114, wherein the cancer isselected from the group consisting of: solid tumor, hematological tumor,sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-celllymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lungcancer, renal cell carcinoma, gastic and esophageal cancer, pancreaticcancer, prostate cancer, breast cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, squamous cell head and neckcarcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma.

Embodiment A116. The method of embodiment A113, wherein the subject hasbeen identified or diagnosed as having an aging-related disease orcondition.

Embodiment A117. The method of embodiment A116, wherein theaging-related disease or condition is selected from the group consistingof: Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpanceatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction.

Embodiment A118. The method of embodiment A113, wherein the subject hasbeen diagnosed or identified as having an infectious disease.

Embodiment A119. The method of embodiment A118, wherein the infectiousdisease is infection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment A120. Nucleic acid encoding any of the single-chain chimericpolypeptides of any one of embodiments A1-A59.

Embodiment A121. A vector comprising the nucleic acid of embodimentA120.

Embodiment A122. The vector of embodiment A121, wherein the vector is anexpression vector.

Embodiment A123. A cell comprising the nucleic acid of embodiment A120or the vector of embodiment A121 or A122.

Embodiment A124. A method of producing a single-chain chimericpolypeptide, the method comprising:

culturing the cell of embodiment A123 in a culture medium underconditions sufficient to result in the production of the single-chainchimeric polypeptide; and

recovering the single-chain chimeric polypeptide from the cell and/orthe culture medium.

Embodiment A125. A single-chain chimeric polypeptide produced by themethod of embodiment A124.

Embodiment A126. The single-chain chimeric polypeptide of embodimentA26, wherein the soluble human tissue factor domain comprises a sequencethat is at least 80% identical to SEQ ID NO: 96.

Embodiment A127. The single-chain chimeric polypeptide of embodimentA126, wherein the soluble human tissue factor domain comprises asequence that is at least 90% identical to SEQ ID NO: 96.

Embodiment A128. The single-chain chimeric polypeptide of embodimentA127, wherein the soluble human tissue factor domain comprises asequence that is at least 95% identical to SEQ ID NO: 96.

Embodiment A129. The single-chain chimeric polypeptide of embodimentA128, wherein the soluble human tissue factor domain comprises asequence that is 100% identical to SEQ ID NO: 96.

Embodiment A130. The single-chain chimeric polypeptide of embodimentA26, wherein the soluble human tissue factor domain comprises a sequencethat is at least 80% identical to SEQ ID NO: 97.

Embodiment A131. The single-chain chimeric polypeptide of embodimentA130, wherein the soluble human tissue factor domain comprises asequence that is at least 90% identical to SEQ ID NO: 97.

Embodiment A132. The single-chain chimeric polypeptide of embodimentA131, wherein the soluble human tissue factor domain comprises asequence that is at least 95% identical to SEQ ID NO: 97.

Embodiment A133. The single-chain chimeric polypeptide of embodimentA132, wherein the soluble human tissue factor domain comprises asequence that is 100% identical to SEQ ID NO: 97.

B. EXEMPLARY EMBODIMENTS

Embodiment B1. A single-chain chimeric polypeptide comprising:

-   -   (i) a first target-binding domain;    -   (ii) a soluble tissue factor domain; and    -   (iii) a second target-binding domain,

wherein:

the first target-binding domain and the second target-binding domaineach specifically bind to an IL-2 receptor; or

the first target-binding domain and the second target-binding domaineach specifically bind to an IL-15 receptor.

Embodiment B2. The single-chain chimeric polypeptide of embodiment B1,wherein the first target-binding domain and the soluble tissue factordomain directly abut each other.

Embodiment B3. The single-chain chimeric polypeptide of embodiment B1,wherein the single-chain chimeric polypeptide further comprises a linkersequence between the first target-binding domain and the soluble tissuefactor domain.

Embodiment B4. The single-chain chimeric polypeptide of any one ofembodiments B1-B3, wherein the soluble tissue factor domain and thesecond target-binding domain directly abut each other.

Embodiment B5. The single-chain chimeric polypeptide of any one ofembodiments B1-B3, wherein the single-chain chimeric polypeptide furthercomprises a linker sequence between the soluble tissue factor domain andthe second target-binding domain.

Embodiment B6. The single-chain chimeric polypeptide of embodiment B1,wherein the first target-binding domain and the second target-bindingdomain directly abut each other.

Embodiment B7. The single-chain chimeric polypeptide of embodiment B1,wherein the single-chain chimeric polypeptide further comprises a linkersequence between the first target-binding domain and the secondtarget-binding domain.

Embodiment B8. The single-chain chimeric polypeptide of embodiment B6 orB7, wherein the second target-binding domain and the soluble tissuefactor domain directly abut each other.

Embodiment B9. The single-chain chimeric polypeptide of embodiment B6 orB7, wherein the single-chain chimeric polypeptide further comprises alinker sequence between the second target-binding domain and the solubletissue factor domain.

Embodiment B10. The single-chain chimeric polypeptide of any one ofembodiments B1-B9, wherein both the first target-binding domain and thesecond target-binding domain is a soluble interleukin protein.

Embodiment B11. The single-chain chimeric polypeptide of embodiment B10,wherein the first target-binding domain and the second target-bindingdomain is a soluble IL-2 protein.

Embodiment B12. The single-chain chimeric polypeptide of embodiment B11,wherein the soluble IL-2 protein is a soluble human IL-2 protein.

Embodiment B13. The single-chain chimeric polypeptide of embodiment B12,wherein the soluble human IL-2 protein comprises SEQ ID NO: 28.

Embodiment B14. The single-chain chimeric polypeptide of embodiment B10,wherein the first target-binding domain and the second target-bindingdomain is a soluble IL-15 protein.

Embodiment B15. The single-chain chimeric polypeptide of embodiment B14,wherein the soluble IL-15 protein is a soluble human IL-15 protein.

Embodiment B16. The single-chain chimeric polypeptide of embodiment B15,wherein the soluble human IL-15 protein comprises SEQ ID NO: 39.

Embodiment B17. The single-chain chimeric polypeptide of any one ofembodiments B1-B16, wherein the soluble tissue factor domain is asoluble human tissue factor domain.

Embodiment B18. The single-chain chimeric polypeptide of embodiment B17,wherein the soluble human tissue factor domain comprises a sequence thatis at least 80% identical to SEQ ID NO: 9.

Embodiment B19. The single-chain chimeric polypeptide of embodiment B18,wherein the soluble human tissue factor domain comprises a sequence thatis at least 90% identical to SEQ ID NO: 9.

Embodiment B20. The single-chain chimeric polypeptide of embodiment B19,wherein the soluble human tissue factor domain comprises a sequence thatis at least 95% identical to SEQ ID NO: 9.

Embodiment B21. The single-chain chimeric polypeptide of any one ofembodiments B17-B20, wherein the soluble human tissue factor domain doesnot comprise one or more of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment B22. The single-chain chimeric polypeptide of embodiment B21,wherein the soluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment B23. The single-chain chimeric polypeptide of any one ofembodiments B1-B22, wherein the soluble tissue factor domain is notcapable of binding Factor VIIa.

Embodiment B24. The single-chain chimeric polypeptide of any one ofembodiments B1-B23, wherein the soluble tissue factor domain does notconvert inactive Factor X into Factor Xa.

Embodiment B25. The single-chain chimeric polypeptide of any one ofembodiments B1-B24, wherein the single-chain chimeric polypeptide doesnot blood stimulate coagulation in a mammal.

Embodiment B26. The single-chain chimeric polypeptide of any one ofembodiments B1-B25, wherein the single-chain chimeric polypeptidefurther comprises one or more additional target-binding domains at itsN- and/or C-terminus.

Embodiment B27. The single-chain chimeric polypeptide of embodiment B26,wherein the single-chain chimeric polypeptide comprises one or moreadditional target-binding domains at its N-terminus.

Embodiment B28. The single-chain chimeric polypeptide of embodiment B27,wherein one or more additional target-binding domains directly abuts thefirst target-binding domain, the second target-binding domain, or thesoluble tissue factor domain.

Embodiment B29. The single-chain chimeric polypeptide of embodiment B28,wherein the single-chain chimeric polypeptide further comprises a linkersequence between one of the at least one additional target-bindingdomains and the first target-binding domain, the second target-bindingdomain, or the soluble tissue factor domain.

Embodiment B30. The single-chain chimeric polypeptide of embodiment B26,wherein the single-chain chimeric polypeptide comprises one or moreadditional target-binding domains at its C-terminus.

Embodiment B31. The single-chain chimeric polypeptide of embodiment B30,wherein one of the one or more additional target-binding domainsdirectly abuts the first target-binding domain, the secondtarget-binding domain, or the soluble tissue factor domain.

Embodiment B32. The single-chain chimeric polypeptide of embodiment B30,wherein the single-chain chimeric polypeptide further comprises a linkersequence between one of the at least one additional target-bindingdomains and the first target-binding domain, the second target-bindingdomain, or the soluble tissue factor domain.

Embodiment B33. The single-chain chimeric polypeptide of embodiment B26,wherein the single-chain chimeric polypeptide comprises one or moreadditional target binding domains at its N-terminus and the C-terminus.

Embodiment B34. The single-chain chimeric polypeptide of embodiment B33,wherein one of the one or more additional antigen binding domains at theN-terminus directly abuts the first target-binding domain, the secondtarget-binding domain, or the soluble tissue factor domain.

Embodiment B35. The single-chain chimeric polypeptide of embodiment B33,wherein the single-chain chimeric polypeptide further comprises a linkersequence between one of the one or more additional antigen-bindingdomains at the N-terminus and the first target-binding domain, thesecond target-binding domain, or the soluble tissue factor domain.

Embodiment B36. The single-chain chimeric polypeptide of embodiment B33,wherein one of the one or more additional antigen binding domains at theC-terminus directly abuts the first target-binding domain, the secondtarget-binding domain, or the soluble tissue factor domain.

Embodiment B37. The single-chain chimeric polypeptide of embodiment B33,wherein the single-chain chimeric polypeptide further comprises a linkersequence between one of the one or more additional antigen-bindingdomains at the C-terminus and the first target-binding domain, thesecond target-binding domain, or the soluble tissue factor domain.

Embodiment B38. The single-chain chimeric polypeptide of any one ofembodiments B26-B37, wherein each of the first target-binding domain,the second target-binding domain, and the one or more additionaltarget-binding domains bind specifically to an IL-2 receptor or an IL-15receptor.

Embodiment B39. The single-chain chimeric polypeptide of embodiment B38,wherein each of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains comprise the same amino acid sequence.

Embodiment B40. The single-chain chimeric polypeptide of any one ofembodiments B26-B37, wherein the one or more additional target-bindingdomains is an antigen-binding domain.

Embodiment B41. The single-chain chimeric polypeptide of embodiment B40,wherein the antigen-binding domain comprises a scFv or a single domainantibody.

Embodiment B42. The single-chain chimeric polypeptide of any one ofembodiments B26-B37, B40, and B41, wherein the one or more additionaltarget-binding domains bind specifically to a target selected from thegroup consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123,IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4,MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R,MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3,EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4,TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-β receptor II(TGF-βRII), a ligand of TGF-βRIII, a ligand of DNAM-1, a ligand ofNKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKp30, a ligandfor a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptorfor IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7,a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, areceptor for IL-15, a receptor for IL-17, a receptor for IL-18, areceptor for IL-21, a receptor for PDGF-DD, a receptor for stem cellfactor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand(FLT3L), a receptor for MICA, a receptor for MICB, a receptor for aULP16-binding protein, a receptor for CD155, a receptor for CD122, and areceptor for CD28.

Embodiment B43. The single-chain chimeric polypeptide of any one ofembodiments B6-B37, B40, and B41, wherein the one or more additionaltarget-binding domains is a soluble interleukin or cytokine protein.

Embodiment B44. The single-chain chimeric polypeptide of embodiment B43,wherein the soluble interleukin or cytokine protein is selected from thegroup consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, IL-21, PDGF-DD, and SCF.

Embodiment B45. The single-chain chimeric polypeptide of any one ofembodiments B6-B37, B40, and B41, wherein the one or more additionaltarget-binding domains is a soluble interleukin or cytokine receptor.

Embodiment B46. The single-chain chimeric polypeptide of embodiment B45,wherein the soluble receptor is a soluble TGF-β receptor II (TGF-βRII)and a soluble TGF-βRIII.

Embodiment B47. The single-chain chimeric polypeptide of any one ofembodiments B1-B46, wherein the single-chain chimeric polypeptidefurther comprises a signal sequence at its N-terminal end.

Embodiment B48. The single-chain chimeric polypeptide of any one ofembodiments B1-B47, wherein the single-chain chimeric polypeptidefurther comprises a peptide tag positioned at the N-terminal end or theC-terminal end of the single-chain chimeric polypeptide.

Embodiment B49. A composition comprising any of the single-chainchimeric polypeptides of embodiments B1-B48.

Embodiment B50. The composition of embodiment B49, wherein thecomposition is a pharmaceutical composition.

Embodiment B51. A kit comprising at least one dose of the composition ofembodiment B49 or B50.

Embodiment B52. A method of stimulating an immune cell, the methodcomprising:

contacting an immune cell with an effective amount of any of thesingle-chain chimeric polypeptides of embodiments B1-B48 or thecomposition of embodiment B49 or B50.

Embodiment B53. The method of embodiment B52, wherein the immune cell iscontacted in vitro.

Embodiment B54. The method of embodiment B53, wherein the immune cellwas previously obtained from a subject.

Embodiment B55. The method of embodiment B54, wherein the method furthercomprises obtaining the immune cell from the subject prior to thecontacting step.

Embodiment B56. The method of embodiment B52, wherein the immune cell iscontacted in vivo.

Embodiment B57. The method of any one of embodiments B52-B56, whereinthe immune cell is selected from the group consisting of: an immaturethymocyte, a peripheral blood lymphocyte, a naïve T cell, a pluripotentTh cell precursor, a lymphoid progenitor cell, a Treg cell, a memory Tcell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, aTh3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ Tcell, a CD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage,a neutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment B58. The method of any one of embodiments B52-B57, whereinthe immune cell has previously been genetically modified to express achimeric antigen receptor or a recombinant T-cell receptor.

Embodiment B59. The method of any one of embodiments B52-B57, whereinthe method further comprises, after the contacting step, introducinginto the immune cell a nucleic acid encoding a chimeric antigen-receptoror a recombinant T-cell receptor.

Embodiment B60. The method of any one of embodiments B52-B59, whereinthe method further comprises administering the immune cell to a subjectin need thereof.

Embodiment B61. The method of embodiment B60, wherein the subject hasbeen identified or diagnosed as having an age-related disease orcondition.

Embodiment B62. The method of embodiment B61, wherein the age-relateddisease or condition is selected from the group consisting of:Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction.

Embodiment B63. The method of embodiment B60, wherein the subject hasbeen identified or diagnosed as having a cancer.

Embodiment B64. The method of embodiment B63, wherein the cancer isselected from the group consisting of: solid tumor, hematological tumor,sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-celllymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lungcancer, renal cell carcinoma, gastric and esophageal cancer, pancreaticcancer, prostate cancer, breast cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, squamous cell head and neckcarcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma.

Embodiment B65. The method of embodiment B60, wherein the subject hasbeen diagnosed or identified as having an infectious disease.

Embodiment B66. The method of embodiment B65, wherein the infectiousdisease is infection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment B67. A method of inducing or increasing proliferation of animmune cell, the method comprising:

contacting an immune cell with an effective amount of any of thesingle-chain chimeric polypeptides of embodiments B1-B48 or thecomposition of embodiment B49 or B50.

Embodiment B68. The method of embodiment B67, wherein the immune cell iscontacted in vitro.

Embodiment B69. The method of embodiment B68, wherein the immune cellwas previously obtained from a subject.

Embodiment B70. The method of embodiment B60, wherein the method furthercomprises obtaining the immune cell from the subject prior to thecontacting step.

Embodiment B71. The method of embodiment B67, wherein the immune cell iscontacted in vivo.

Embodiment B72. The method of any one of embodiments B67-B71, whereinthe immune cell is selected from the group consisting of: an immaturethymocyte, a peripheral blood lymphocyte, a naïve T cell, a pluripotentTh cell precursor, a lymphoid progenitor cell, a Treg cell, a memory Tcell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, aTh3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ Tcell, a CD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage,a neutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment B73. The method of any one of embodiments B67-B72, whereinthe immune cell has previously been genetically modified to express achimeric antigen receptor or a recombinant T-cell receptor.

Embodiment B74. The method of any one of embodiments B67-B72, whereinthe method further comprises, after the contacting step, introducinginto the immune cell a nucleic acid encoding a chimeric antigen-receptoror a recombinant T-cell receptor.

Embodiment B75. The method of any one of embodiments B67-B74, whereinthe method further comprises administering the immune cell to a subjectin need thereof.

Embodiment B76. The method of embodiment B75, wherein the subject hasbeen identified or diagnosed as having an age-related disease orcondition.

Embodiment B77. The method of embodiment B76, wherein the age-relateddisease or condition is selected from the group consisting of:Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction.

Embodiment B78. The method of embodiment B75, wherein the subject hasbeen identified or diagnosed as having a cancer.

Embodiment B79. The method of embodiment B78, wherein the cancer isselected from the group consisting of: solid tumor, hematological tumor,sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-celllymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lungcancer, renal cell carcinoma, gastric and esophageal cancer, pancreaticcancer, prostate cancer, breast cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, squamous cell head and neckcarcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma.

Embodiment B80. The method of embodiment B75, wherein the subject hasbeen diagnosed or identified as having an infectious disease.

Embodiment B81. The method of embodiment B75, wherein the infectiousdisease is infection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment B82. A method of inducing differentiation of an immune cellinto a memory or memory-like immune cell, the method comprising:

contacting an immune cell with an effective amount of any of thesingle-chain chimeric polypeptides of embodiments B1-B48 or thecomposition of embodiment B49 or B50.

Embodiment B83. The method of embodiment B82, wherein the immune cell iscontacted in vitro.

Embodiment B84. The method of embodiment B83, wherein the immune cellwas previously obtained from a subject.

Embodiment B85. The method of embodiment B84, wherein the method furthercomprises obtaining the immune cell from the subject prior to thecontacting step.

Embodiment B86. The method of embodiment B82, wherein the immune cell iscontacted in vivo.

Embodiment B87. The method of any one of embodiments B82-B86, whereinthe immune cell is selected from the group consisting of: an immaturethymocyte, a peripheral blood lymphocyte, a naïve T cell, a pluripotentTh cell precursor, a lymphoid progenitor cell, a Treg cell, a Th17 cell,a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell, γδ T cell,an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ T cell, a CD4⁺ T cell,a natural killer T cell, a mast cell, a macrophage, a neutrophil, adendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment B88. The method of any one of embodiments B82-B87, whereinthe immune cell has previously been genetically modified to express achimeric antigen receptor or a recombinant T-cell receptor.

Embodiment B89. The method of any one of embodiments B82-B87, whereinthe method further comprises, after the contacting step, introducinginto the immune cell a nucleic acid encoding a chimeric antigen-receptoror a recombinant T-cell receptor.

Embodiment B90. The method of any one of embodiments B82-B89, whereinthe method further comprises administering the immune cell to a subjectin need thereof.

Embodiment B91. The method of embodiment B90, wherein the subject hasbeen identified or diagnosed as having an age-related disease orcondition.

Embodiment B92. The method of embodiment B91, wherein the age-relateddisease or condition is selected from the group consisting of:Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction.

Embodiment B93. The method of embodiment B90, wherein the subject hasbeen identified or diagnosed as having a cancer.

Embodiment B94. The method of embodiment B93, wherein the cancer isselected from the group consisting of: solid tumor, hematological tumor,sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-celllymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lungcancer, renal cell carcinoma, gastric and esophageal cancer, pancreaticcancer, prostate cancer, breast cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, squamous cell head and neckcarcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma.

Embodiment B95. The method of embodiment B90, wherein the subject hasbeen diagnosed or identified as having an infectious disease.

Embodiment B96. The method of embodiment B95, wherein the infectiousdisease is infection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment B97. A method of killing a cancer cell, an infected cell, ora senescent cell in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anyof the single-chain chimeric polypeptides of embodiments B1-B48 or thecomposition of embodiment B49 or B50.

Embodiment B98. The method of embodiment B97, wherein the subject hasbeen identified or diagnosed as having a cancer.

Embodiment B99. The method of embodiment B98, wherein the cancer isselected from the group consisting of: solid tumor, hematological tumor,sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-celllymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lungcancer, renal cell carcinoma, gastric and esophageal cancer, pancreaticcancer, prostate cancer, breast cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, squamous cell head and neckcarcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma.

Embodiment B100. The method of embodiment B97, wherein the subject hasbeen identified or diagnosed as having an aging-related disease orcondition.

Embodiment B101. The method of embodiment B100, wherein theaging-related disease or condition is selected from the group consistingof: Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction.

Embodiment B102. A method of treating a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of any of the single-chain chimeric polypeptides ofembodiments B1-B48 or the composition of embodiment B49 or B50.

Embodiment B103. The method of embodiment B102, wherein the subject hasbeen identified or diagnosed as having a cancer.

Embodiment B104. The method of embodiment B103, wherein the cancer isselected from the group consisting of: solid tumor, hematological tumor,sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma,rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms,multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma,Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-celllymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lungcancer, renal cell carcinoma, gastric and esophageal cancer, pancreaticcancer, prostate cancer, breast cancer, colorectal cancer, ovariancancer, non-small cell lung carcinoma, squamous cell head and neckcarcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma.

Embodiment B105. The method of embodiment B102, wherein the subject hasbeen identified or diagnosed as having an aging-related disease orcondition.

Embodiment B106. The method of embodiment B105, wherein theaging-related disease or condition is selected from the group consistingof: Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction.

Embodiment B107. The method of embodiment B102, wherein the subject hasbeen diagnosed or identified as having an infectious disease.

Embodiment B108. The method of embodiment B107, wherein the infectiousdisease is infection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment B109. A nucleic acid encoding any of the single-chainchimeric polypeptides of any one of embodiments B1-B48.

Embodiment B110. A vector comprising the nucleic acid of embodimentB109.

Embodiment B111. The vector of embodiment B110, wherein the vector is anexpression vector.

Embodiment B112. A cell comprising the nucleic acid of embodiment B109or the vector of embodiment B110 or B111.

Embodiment B113. A method of producing a single-chain chimericpolypeptide, the method comprising:

culturing the cell of embodiment B112 in a culture medium underconditions sufficient to result in the production of the single-chainchimeric polypeptide; and

recovering the single-chain chimeric polypeptide from the cell and/orthe culture medium.

Embodiment B114. A single-chain chimeric polypeptide produced by themethod of embodiment B113.

Embodiment B115. The single-chain chimeric polypeptide of embodimentB21, wherein the soluble human tissue factor domain comprises a sequencethat is at least 80% identical to SEQ ID NO: 96.

Embodiment B116. The single-chain chimeric polypeptide of embodimentB115, wherein the soluble human tissue factor domain comprises asequence that is at least 90% identical to SEQ ID NO: 96.

Embodiment B117. The single-chain chimeric polypeptide of embodimentB116, wherein the soluble human tissue factor domain comprises asequence that is at least 95% identical to SEQ ID NO: 96.

Embodiment B118. The single-chain chimeric polypeptide of embodimentB117, wherein the soluble human tissue factor domain comprises asequence that is 100% identical to SEQ ID NO: 96.

Embodiment B119. The single-chain chimeric polypeptide of embodimentB21, wherein the soluble human tissue factor domain comprises a sequencethat is at least 80% identical to SEQ ID NO: 97.

Embodiment B120. The single-chain chimeric polypeptide of embodimentB119, wherein the soluble human tissue factor domain comprises asequence that is at least 90% identical to SEQ ID NO: 97.

Embodiment B121. The single-chain chimeric polypeptide of embodimentB120, wherein the soluble human tissue factor domain comprises asequence that is at least 95% identical to SEQ ID NO: 97.

Embodiment B122. The single-chain chimeric polypeptide of embodimentB121, wherein the soluble human tissue factor domain comprises asequence that is 100% identical to SEQ ID NO: 97.

What is claimed is:
 1. A method of treating a subject havingatherosclerosis, the method comprising administering to the subject atherapeutically effective amount of a single-chain chimeric polypeptidecomprising: (i) a first target-binding domain; (ii) a soluble tissuefactor domain does not have coagulation activity; and (iii) a secondtarget-binding domain, wherein: the first target-binding domain and thesecond target-binding domain bind to a receptor for IL-2; and thesingle-chain chimeric polypeptides does not stimulate blood coagulationin a mammal.
 2. The method of claim 1, wherein the first target-bindingdomain and the soluble tissue factor domain directly abut each other. 3.The method of claim 1, wherein the single-chain chimeric polypeptidefurther comprises a linker sequence between the first target-bindingdomain and the soluble tissue factor domain.
 4. The method of claim 1,wherein the soluble tissue factor domain and the second target-bindingdomain directly abut each other.
 5. The method of claim 1, wherein thesingle-chain chimeric polypeptide further comprises a linker sequencebetween the soluble tissue factor domain and the second target-bindingdomain.
 6. The method of claim 1, wherein one or both of the firsttarget-binding domain and the second target-binding domain is anantigen-binding domain.
 7. The method of claim 1, wherein one or both ofthe first target-binding domain and the second target-binding domain ishuman IL-2.
 8. The method of claim 7, wherein the first target-bindingdomain and the second target-binding domain are human IL-2.
 9. Themethod of claim 8, wherein the human IL-2 comprises a sequence that isat least 90% identical to SEQ ID NO:
 28. 10. The method of claim 9,wherein the human IL-2 comprises a sequence that is at least 95%identical to SEQ ID NO:
 28. 11. The method of claim 10, wherein thehuman IL-2 comprises SEQ ID NO:
 28. 12. The method of claim 1, whereinthe soluble tissue factor domain is a soluble human tissue factor domainthat does not have coagulation activity.
 13. The method of claim 1,wherein the soluble tissue factor domain comprises or consists of asequence from a wildtype soluble human tissue factor.
 14. The method ofclaim 1, wherein the soluble tissue factor domain comprises a sequencethat is at least 90% identical to SEQ ID NO:
 9. 15. The method of claim14, wherein the soluble tissue factor domain comprises a sequence thatis at least 95% identical to SEQ ID NO:
 9. 16. The method of claim 15,wherein the soluble tissue factor domain comprises SEQ ID NO:
 9. 17. Themethod of claim 1, wherein the single-chain chimeric polypeptidecomprises a sequence that is at least 90% identical to SEQ ID NO: 108.18. The method of claim 17, wherein the single-chain chimericpolypeptide comprises a sequence that is at least 95% identical to SEQID NO:
 108. 19. The method of claim 18, wherein the single-chainchimeric polypeptide comprises SEQ ID NO:
 108. 20. The method of claim1, wherein the single-chain chimeric polypeptide comprises a sequencethat is at least 90% identical to SEQ ID NO:
 110. 21. The method ofclaim 20, wherein the single-chain chimeric polypeptide comprises asequence that is at least 95% identical to SEQ ID NO:
 110. 22. Themethod of claim 21, wherein the single-chain chimeric polypeptidecomprises SEQ ID NO: 110.